Il-23r antagonists to reprogram intratumoral t regulatory cells into effector cells

ABSTRACT

Provided by the disclosure are compositions and methods for modulating differentiation of regulatory T cells. In some embodiments, methods include selectively decreasing IL-23R activity and/or IL-23R expression without significantly decreasing IL-12RP activity and/or IL-12RP expression.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 62/500,443, filed on May 2, 2017,entitled “IL-23R ANTAGONISTS TO REPROGRAM INTRATUMORAL T REGULATORYCELLS INTO EFFECTOR CELLS,” the entire contents of which areincorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01AI37562 and5T32AI074549-07 awarded by the National Institute of Allergy andInfectious Diseases. The government has certain rights in the invention.

BACKGROUND OF INVENTION

Regulatory T cells (Treg cells) are critically important for maintenanceof a balanced immune system, in part because they help to dampenexcessive immune responses and prevent autoimmunity. However, immunesuppression exerted by Treg cells can impede anti-tumor immune responsesand promote cancer growth. The efficacy of several successfulimmunotherapies has been associated with depletion or blockade of CD4Treg cells (Mahoney et al., Nat Rev Drug Discov 14:561-584).

SUMMARY OF INVENTION

The disclosure relates to selective induction of Treg cell phenotypicinstability and selective conversion of intratumoroal, but not systemic,Treg cells into T effector cells (Teff cells). The disclosure relates tocompositions for modulating the differentiation of Treg cells. Thedisclosure is based, in part, on the surprising discovery that theIL-23R cytokine receptor subunit is expressed on Treg cells and thatinhibition of IL-23R induces differentiation of Treg cells (e.g., toTeff cells). Inducing Treg cell differentiation is particularly useful,for example, in the context of treating cancer.

In one aspect, disclosed herein are methods for treating cancer in ahuman subject. The methods involve administering to a human subject inneed thereof an agent that decreases IL-23R activity and does notdecrease IL-12Rβ activity, in an amount effective to treat the cancer.In embodiments, the agent is a polypeptide, nucleic acid, or smallmolecule. In some embodiments, the agent binds the extracellular domainof IL-23R.

In some embodiments, the agent is a human or humanized monoclonalantibody. In some embodiments, the antibody binds IL-23R and does notbind IL-12Rβ. In some embodiments, the antibody has 100-fold,1,000-fold, or 10,000-fold greater binding affinity for IL-23R than forIL-12Rβ. In some embodiments, the antibody is free of antibody-dependentcellular cytotoxicity (ADCC) activity. In some embodiments, the antibodyhas an IgG4 heavy chain immunoglobulin constant domain.

In some embodiments, the agent binds a nucleic acid coding for orexpressing IL-23R.

In some embodiments, the methods further comprise administering to thehuman subject an immunomodulatory agent. In some embodiments, theimmunomodulatory agent is an immune checkpoint inhibitor. In someembodiments, immune checkpoint inhibitor is an antagonist of a moleculeselected from the group consisting of PD-1, TIM-3, TIGIT, VISTA, A2AR,B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. In some embodiments, theimmunomodulatory agent in an antagonist of inhibitory macrophages and/ordendritic cells.

In some embodiments, the method further comprises administering an agentthat inhibits cancer cell growth.

In another aspect, disclosed herein are methods for inducingdifferentiation of a Treg cell to a Teff cell. The methods involvecontacting the Treg cell in vitro with an agent that decreases IL-23Ractivity and does not decrease IL-12Rβ activity. In some embodiments,the agent is a polypeptide, nucleic acid, or small molecule.

In some embodiments, the agent binds the extracellular domain of IL-23R.In some embodiments, the agent is a human or humanized monoclonalantibody. In some embodiments, the antibody binds IL-23R and does notbind IL-12Rβ. In some embodiments, the antibody has 100-fold,1,000-fold, or 10,000-fold greater binding affinity for IL-23R than forIL-12Rβ. In some embodiments, the antibody is free of antibody-dependentcellular cytotoxicity (ADCC) activity. In some embodiments, the antibodyhas an IgG4 heavy chain immunoglobulin constant domain.

In some embodiments, the agent binds a nucleic acid coding for orexpressing IL-23R.

In some embodiments, the Treg cell is a CD4+ Treg cell, a CD8+ Tregcell, a Helios+ Treg cell, or a CD25+ and FoxP3+ Treg cell.

In some embodiments, the method further comprises contacting the Tregcell in the presence of one or more inflammatory cytokines. In someembodiments, the one or more inflammatory cytokines is selected from thegroup consisting of IL-2, IL-4, and combination thereof.

In another aspect, disclosed herein are methods for identifying acompound that will convert a Treg cell into a Teff cell. The methodscomprise:

-   -   contacting a Treg cell with a test compound;    -   measuring IL-23R activity in the cell; and    -   measuring IL-12Rβ activity in the cell;    -   wherein the test compound is a compound for converting a Treg        cell into an effector cell if:        (i) the IL-23R activity is decreased relative to a control cell        that has been treated with a compound known to not decrease        IL-23R activity and IL-12Rβ activity; and        (ii) the IL-12Rβ activity is not decreased relative to the        control cell.

In some embodiments, the methods further comprise measuring FoxP3activity in the cell.

In some embodiments, the methods further comprise measuring an effectorcytokine produced by the cell. In some embodiments, the effectorcytokine is TNF-α, IFN-γ, IL-17, IL-10, or IL-2.

In some embodiments, the test compound is selected from the groupconsisting of a polypeptide, a nucleic acid and a small molecule.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIGS. 1A-1B show that Treg specific Helios ^(−/−) mice exhibit elevatedantitumor immunity. Treg specific Helios ^(+/+) and Helios ^(−/−) mice(Helios^(WT) and Helios^(KO) respectively) were inoculated with B16/F10or MC38 cancer cell lines and assessed for tumor progression andcytotoxic T cell activity. FIG. 1A shows tumor volume (mm³) ofsubcutaneously injected B16/F10 or MC38 cells. FIG. 1B showsintratumoral CD8 and IFN-γ expression. Representative FACS plots fromtriplicate experiments are shown. P value: *<0.05.

FIGS. 2A-2B. show Helios binding motifs in the first intron of STAT5busing ChiP-Seq analysis of Helios binding and modified histones atSTAT5b in CD4 nTregs. The start sites of each gene locus are indicated.The vertical lines in the gene diagrams (bottom) indicate exons. FIG. 2Ashows the Helios binding site in the first intron of the STAT5b genelocus. FIG. 2B shows the Helios binding motifs from genome wide analysisof Helios binding sites.

FIGS. 3A-3C show isolated function of Helios deficient CD4 Treg inantitumor immunity. nTregs were isolated from Rag2^(−/−) mice (2×10⁶CD4: 1×10⁶ CD8: 0.5×10⁶ Helios^(WT) or Helios^(KO) nTreg) B16/F10 tumor.Flow cytometry was gated on CD45.2⁺FoxP3⁺ cells. FIG. 3A shows theB16/F10 tumor growth (volume mm³) curve beginning with first appearanceof tumor (day 10). FIG. 3B shows the proportion of splenic and tumorinfiltrating nTregs positive for FoxP3 expression. FIG. 3C shows theproportion of splenic and tumor infiltrating nTregs positive for INF-γexpression. Representative FACS plots from triplicate experiments areshown.

FIGS. 4A-4B show that Treg specific Helios^(KO) mice present a reducedanergic phenotype. Anergic phenotype was determined in Treg specificHelios^(WT) and Helios^(KO) spleen and tumor infiltrate (MC38 inoculatedmice) using co-expression of surface markers CD73 and FR4 in FoxP3⁺Treg. FIG. 4A shows expression of FoxP3 and FIG. 4B shows expression ofCD73^(high)FR4^(high) cells. Both FIGS. 4A-4B show representative FACSplots from triplicate experiments. P value: *<0.05, **, <0.01.

FIGS. 5A-5B show that Helios⁺ nTreg lose surface receptor expressionco-cultured with IL-4. nTreg were co-cultured with anti-CD3/anti-CD28coated beads as well as steady state cytokine IL-2 and inflammatorycytokine IL-4. FIG. 5A shows a loss of Helios expression upon additionof IL-4. FIG. 5B shows the dose dependent IL-4-mediated shift inHelios⁺TIGIT⁺ nTreg proportion. The cells were gated onTCR⁺CD4⁺CD25⁺FoxP3⁺.

FIG. 6 shows a relevant Treg pathways in the inflammatory tumormicroenvironment. Treg stabilize their suppressive phenotype by 1)enhanced IL-2 responsiveness via a Helios dependent genetic programand/or 2) enhanced STAT5 activation by IL-23R signaling that is uniquelyoperative within the tumor microenvironment. The blockade of eithersignaling pathway induces Treg conversion.

FIGS. 7A-7D shows that ligation of specific interleukin receptorsinduces Treg conversion in vitro. Receptors of interest were screenedfor loss of FoxP3 (FIG. 7A) and gain on IFN-γ expression (FIG. 7B).Anti-IL-23R, which resulted in the highest IFN-γ expression, wasselected for further analysis. The kinetics of anti-IL-23R ligation onTreg conversion was assessed at 24 and 120 hours (5 days). A significantdecrease in FoxP3 (FIG. 7C) and subsequent increase in IFN-γ (FIG. 7D)was observed when anti-IL-23R was co-cultured with Treg after 24 hoursof stimulation was observed. ANOVA:*p<0.5**p<0.01***p<0.001****p<0.0001.

FIG. 8 shows that FoxP3 and IFN-γ expression after engagement of GITRinduces Helios downregulation in CD4 Treg. FoxP3⁺ CD4 Treg were isolatedfrom spleens of wild type B6 mice and cultured in the anti-CD3/CD28coated wells in the presence of IL-4, increasing doses of IL-2, andanti-GITR (DTA-1) or isotype antibodies. After 5 days, the cells wereanalyzed for the levels of FoxP3 expression and IFN-γ production.

FIGS. 9A-9B provide confirmation of the in vitro conversion assay usinga known TNF receptor super family member GITR agonist. Sorted C57BL/6(WT) Treg (>90% pure CD4⁺CD25⁺) were cultured with plate boundanti-CD3/anti-CD28 in the presence of IL-2 and IL-4 for 24 hours or 5days, and then assessed for conversion defined as loss of FoxP3 and gainof IFN-γ expression. The representative dot plots for Treg (gated onTCR⁺CD4⁺ cells) (FIG. 9A) and IFN-γ⁺ cells (gated on FoxP3⁺ cells) (FIG.9B) with graphical representation of change from control (no antibody)are shown. ANOVA: **p≤0.01****p≤0.0001.

FIGS. 10A-10C show that treatment with GITR agonist induces Tregconversion in TME. Wild type (WT) mice were inoculated with B16/F10subcutaneously and treated with an intraperitoneal injectionprophylactically on days 3, 6 and 9 or therapeutically on days 10, 12,14 and 16). FIG. 10A shows tumor growth curves. Intratumoral and splenicTreg were assessed for IFN-γ (gated on FoxP3⁺ cells) (FIG. 10B) andFoxP3 (gated on CD4⁺CD25⁺ cells) (FIG. 10C) expression. Student's Ttest: ***p≤0.001.

FIGS. 11A-11B show in vitro screen results for Treg conversion byanti-IL-23R antibody. Naïve peripheral Treg were sorted at >90% purityand cultured for 5 days in the presence of plate bound anti-CD3 andanti-CD28 as well as soluble IL-2 (20 ng/mL) and IL-4 (50 ng/mL). FIG.11A shows the expression of FoxP3 by Treg (% reduction) and FIG. 11Bshows the fold-increase IFN-γ⁺ Treg cells after co-culture with IL-23associated large molecules. Average±SEM-ANOVA: ***p<0.001***p<0.0001.

FIG. 12 shows FoxP3 and CD25 expression in sorted Treg plated with platebound anti-CD3/anti-CD28 and cultured for 5 days in the presence of IL-2(20 ng/mL) and IL-4 (50 ng/mL). Antibodies were added at 10 ng/mL on Day0 of culture.

FIG. 13 shows that FoxP3^(hi) expression is lost with anti-IL-23R doseresponse. Sorted Treg were plated with plate bound anti-CD3/anti-CD28and cultured for 5 days. In the presence of IL2 (20 ng/mL) and IL4 (50ng/mL), anti-IL-23R antibodies were added at 1, 10 and 25 ng/mLincremental doses on Day 0 of culture. The reduced ratio of FoxP3^(hi)to FoxP3^(lo) Treg is seen in the positive control (anti-GITR [DTA-1] 10ng/mL) as well as in the anti-IL-23R treated cells.

FIG. 14 provides a schematic illustration of the IL-23 receptor complex.The IL-23 cytokine consisting of p19 and p40 chains, binds to theheterodimeric receptor IL-23R and IL-12Rβ.

FIG. 15 shows in vivo intratumoral Treg conversion and IL-23Rexpression. Mice were inoculated with B16/F10 cancer cells and assessedfor IL-23R expression in splenic (left) and tumor infiltrating (right)Treg populations.

FIG. 16 shows IL-23R expression in tumor dLN and tumor infiltrating Tregcells. IL-23R expression is significantly increased in Treg found in thetumor and tumor draining lymph node (TdLN) compared to the spleen. MC38cells were subcutaneously inoculated in C57BL/6 mice. Measurements weremade (no treatment) on day 25 after inoculation. The representative FACsdot plots gated on TCR⁺CD4⁺CD25⁺FoxP3⁺ cells with graphical analysis areshown [Mean±SEM ANOVA ***p>0.001****p.0.0001].

FIG. 17 shows that effector Treg (eTr) express high levels of IL-23R.Intratumoral effector Treg (eTr) have significantly increased IL-23Rexpression compared to TI central Treg (cTr) and splenic Treg. MC38cells were subcutaneously inoculated in C57BL/6. Measurements were madeon day 25 after inoculation. Representative FACs dot plot gated onTCR⁺CD4⁺CD25⁺FoxP3⁺ cells with graphical analysis are shown.

FIG. 18 shows that tumor infiltrating CD69⁺Icos⁺Treg express high levelsof IL-23R. Double positive CD69⁺Icos⁺expression in tumor infiltrating(TI) Treg is significantly increased compared to TdLN and Spleen (SP) inMC38 inoculated mice. Representative FACs dot plots gated onTCR⁺CD4⁺CD25⁺FoxP3⁺ cells with graphical analysis are shown [Mean±SEMANOVA: * p>0.05**p>0.01].

FIG. 19 shows that Helios⁺ Treg express high levels of IL-23R. Helios⁺TI Treg have significantly increased IL-23R expression compared toHelios⁻ TI Treg. Representative FACs dot plots gated onTCR⁺CD4⁺CD25⁺FoxP3⁺ cells with graphical analysis are shown[Mean±SEMStudent's T test: **p>0.01].

FIG. 20 shows that Helios^(+WT) tumor infiltrating Treg havesignificantly increased IL-23R expression in CD69+ Treg compared toHelios^(−WT) and Helios^(KO) TI Treg. Helios^(f/f) (WT) and Helios^(f/f)FoxP3^(cre) (KO) mice were inoculated subcutaneously with F10/B16collected on day 21. The graphical analysis gated on TCR⁺CD4⁺CD25⁺FoxP3⁺for IL-23R expression (top), Treg subsets including CD69+ eTr and cTr(middle), and IL-23R expression in Treg subsets (bottom) are shown[Mean±SEM ANOVA or Student's T test: **p>0.01].

FIG. 21 shows that IL-23 regulates expression of FoxP3 and IFN-γ. SortedTreg (>90% pure CD3⁺CD4⁺CD25⁺) were plated with plate boundanti-CD3/anti-CD28 (10 μg/mL in 1×PBS) and in the presence of IL2 (20ng/mL) and IL4 (50 ng/mL) and cultured for 5 days (120 hours). IL-23recombinant protein was added at 25 ng/mL time 0 (T0) of culture andp(40)₂ was added for the last 24 hours of culture (T96). Representativedot plots are shown for decreased expression of FoxP3 (top left) andincreased expression of IFN-γ (bottom left). The graphicalrepresentation (right) of the fold change from baseline (negativecontrol or no antibody) is shown as mean±SEM. ANOVA: p<0.05.

FIG. 22 shows CD25 expression in sorted Treg plated with plate boundanti-CD3/anti-CD28 and cultured for 5 days in the presence of IL2 (20ng/mL) and IL4 (50 ng/mL). Antibodies and p40 homodimer were added at 0and 96 hours and then cultured for 1 or 5 days.

FIG. 23 shows IFN-γ expression and fractions of IFN-γ expressing cellsin sorted Treg plated with plate bound anti-CD3/anti-CD28 and culturedfor 5 days in the presence of IL2 (20 ng/mL) and IL4 (50 ng/mL).Antibodies and p40 homodimer were added at 0 and 96 hours and thencultured for 1 or 5 days.

FIG. 24 shows tumor outgrowth in control (1×PBS) and p40 homodimertreated mice. Treatment (50 μg/mouse using an intraperitoneal injection)control (n=3) or p40 homodimer (n=4) was given on day 0, 4, and 8 asindicated by the arrows (↓). The tumor volume (mm³) is represented as anaverage (left) and as individual values (right).

FIGS. 25A-25E show in vivo effects of anti-IL-23R treatment. FIG. 25Ashows the protocol for the in vivo experiment. FIGS. 25B-25C show tumoroutgrowth curves. FIG. 25B shows the individual growth curves. Thenumber in the bottom corner indicates how many mice developedtumors/total sample size (day 21). FIG. 25C illustrates the mean tumoroutgrowth±SD. Student's T Test (day 21). FIG. 25D shows that anti-IL-23Rantibody treatment delays tumor outgrowth. Tumor outgrowth in isotypeantibody and anti-IL-23R antibody treated mice is shown. Treatment (100mg/mouse using an intraperitoneal injection) isotype (n=3) anti-IL-23R(n=3) was given on day 8, 11, and 14 as indicated by the arrows (↓). Thetumor volume (mm³) is represented as an average (left) and as individualvalues (right). Student's T test (day 16 data points). FIG. 25E showsthe tumor out growth curve treatment given intraperitoneally (100μg/mouse) on day 8, 11 and 14.

FIGS. 26A-26B show that anti-IL-23R induces intratumoral Treg conversionand increased antitumor activity. FIG. 26A shows CD8 activity as shownby IFN-γ expression. (Representative FACs plots gated on TCR⁺CD8⁺ cellsare shown. FIG. 26B shows intratumoral Treg conversion as indicated byIFN-γ expression Representative FACs plots gated on TCR⁺CD4⁺CD25⁺FoxP3⁺cells are shown. ANOVA or Student's T test:*p<0.05***p<0.001****p<0.0001.

FIG. 27 shows that IL-23R expression is significantly decreased inanti-IL-23R antibody treated tumor and in tumor infiltrating activatedTreg. Representative FACs dot plot gating strategy on single cellslymphocytes to aTreg (top left to right), graphical analysis of IL-23R⁺intratumoral cell proportion (bottom left), and IL-23R⁺aTreg numbers(bottom right) collected on day 21 are shown [Mean±SEM Student's t test:*p>0.05**p.0.01].

FIG. 28 shows that IL-23R expression is significantly decreased in eTrfound in the tumor of anti-IL-23r antibody treated mice. RepresentativeFACs dot plots gated on TCR⁺CD4⁺CD25⁺FoxP3⁺ cells with graphicalanalysis of IL-23r⁺ eTr and cTr collected on day 21 are shown [Mean±SEMStudent's t test *p>0.05].

FIG. 29 shows that anti-IL-23R tumor infiltrating Treg have decreasedHelios expression. Representative dot plots with graphical analysisgated on IL-23R⁺TCR⁺CD4⁺FoxP3⁺ for Helios expression (left) andgraphical analysis (right) are shown [Mean±SEM Student's T test*p>0.05].

FIG. 30 shows that anti-IL-23R treated mice have significantly reducedintratumoral suppressive Treg compared to isotype. The representativedot plots with graphical analysis gated on TCR⁺CD4⁺CD25⁺FoxP3 and IFN-γexpression with graphical analysis on number of tumor infiltrating (TI)Treg, FoxP3^(hi), and IFN-γ expression are shown [Mean±SEM Student's Ttest *p>0.05**p>0.01****p>0.0001].

FIG. 31 shows that anti-IL-23R antibody treatment increases intratumoralCD8/Treg ratio and CD8 IFN-γ expression. Representative FACs dot plotsgated on single cells lymphocytes (left) and TCR⁺CD4⁺CD25⁺FoxP3⁺ (right)cells with graphical analysis are shown [Mean±SEM Student's t test*p>0.05].

FIG. 32 provides a schema for a Treg conversion screening protocol usinga FoxP3^(RFP) IFN-γ^(YFP) reporter system.

FIG. 33 shows tumor volume in mice inoculated with MC38 cells andtreated with isotype or 12B2B64 or 258018 IL-23R antibodies.

FIGS. 34A-34B show conversion of Tregs upon administration of IL-23Rantibodies. Sorted Treg (WT) were plated for 5 days with plate boundanti-CD3/anti-CD28 and in the presence of IL-2 (20 ng/mL) and IL-4 (50ng/mL) with 10 ng/mL 12B2B64 or 258018 IL-23R antibodies or DTA-1. Allsamples were compared to Negative Control (no antibody). FIG. 34A showspercent change of FOXP3 expression. FIG. 34B shows percent change ofIFN-gamma expression. Graphs show combined data from three experiments(mean±SEM ANOVA p<0.05*p<0.01**p<0.001***p<0.0001****.

FIG. 35 shows the eTr phenotype in intratumoral Treg in mice treatedwith IL-23R antibodies. All mice were inoculated with MC38 and treatedwith isotype or 12B2B64 or 258018 IL-23r Ab. Representative dot plotsare gated on Treg (TCR⁺CD4⁺CD25⁺FoxP3⁺) and eTre(TCR⁺CD4⁺CD25⁺FoxP3⁺CD44⁺CD62L⁻). Graphic analysis is based onIL-23r⁺eTr. Mean±SEM **p<0.01.

FIG. 36 shows the pSTAT5:pSTAT3 ratio in mice treated with IL-23Rantibodies. All mice were inoculated with MC38 and treated with isotypeor 12B2B64 or 258018 IL-23r Ab. The STAT5 activation and STAT3activation was measured in tumor and spleen. Representative histogramsare shown as are the pSTAT5:pSTAT3 ratios.

FIG. 37 shows an alignment of murine and human IL-23R and a schematicillustration of the domains of IL-23R.

FIG. 38 shows the regions in the cytokine binding domain of IL-23R wherethe peptides identified in the epitope mapping study are located.

FIGS. 39A-39B show that loss of IL-23R enhances conversion of Treg→Teffgenerally in a Treg conversion assay. FIG. 39A shows FoxP3 expressionlevels and FIG. 39B shows IFN-γ expression levels in IL-23R^(KO) andC57BL/6 Tregs.

FIGS. 40A-40C shows that loss of IL-23R enhances conversion of Treg→Teffin tumor cells. Genetic deletion of IL-23R in Tregs injected intoRag2^(−/−) mice leads to a decrease in tumor volume in mice inoculatedwith the MC38 cancer cell line (FIG. 40A), a decrease in FoxP3expression (FIG. 40B) and an increase in IFN-γ expression (FIG. 40C)compared to IL-23R^(WT) Treg controls. Expression levels from TIL andspleen cells were independently tested.

FIGS. 41A-41B show that anti-IL-23R antibody induces conversion ofTreg→Teff in EL4 lymphoma tumor cells. Treatment of mice inoculated withEL4 cancer cells with anti-IL-23R antibody leads to a decrease in FoxP3expression (FIG. 41A) and an increase in IFN-γ expression (FIG. 41B)compared to treatment with isotype. Expression levels from TIL andspleen cells were independently tested.

FIGS. 42A-42C shows that show that treatment with anti-IL-23R antibodyin vitro alters STAT5 and STAT3 expression. Treatment of CD25 +FoxP3+Tregs with anti-IL-23R antibody leads to a decrease in pSTAT5expression and an increase in pSTAT3 expression compared to treatmentwith isotype (FIG. 42A). These expression changes result in a decreasein the pSTAT5:pSTAT3 ratio for Tregs treated with anti-IL-23R antibodycompared to isotype (FIG. 42B). Treatment with anti-IL-23R antibody alsoleads to increased expression of IFN-γ (FIG. 42C)

FIGS. 43A-43B shows that treatment with anti-IL-23R antibody reducesSTAT5 expression in Treg at multiple timepoints. The reduction in pSTAT5expression resulting in treatment of CD25⁺FoxP3⁺ Tregs with anti-IL-23Rantibody compared to isotype occurs at one hour and persists at fivedays as shown by flow cytometry (FIG. 43A) and as a percentage ofinitial expression (FIG. 43B).

FIGS. 44A-44F shows the effect of an anti-IL-23R antibody treatment inmice that have been inoculated with MC38 cancer cells. FIGS. 44A-44Cshow that treatment with anti-IL-23R antibody provides a reduction inthe pSTAT5:pSTAT3 ratio compared to treatment with isotype. STAT5expression is shown in FIG. 44A and STAT3 expression is shown in FIG.44B. FIG. 44D shows that treatment with anti-IL-23R antibody decreasesthe numbers of CD25⁺FoxP3⁺ Tregs, compared to treatment with isotype.FIGS. 44E-44F show that treatment with anti-IL-23R antibody increasesIFN-γ expression and decreases IL-23R expression levels.

FIGS. 45A-45B show the effect of an anti-IL-23R antibody treatment inmice that have been inoculated with EL4 cancer cells on STAT5 and STAT3expression. Expression of STAT5 in mice inoculated with EL4 cancer cellsand treated with anti-IL-23R antibody is shown in FIG. 45A. ThepSTAT5:pSTAT3 ratio, compared to treatment with isotype, is shown inFIG. 45B.

FIGS. 46A-46C show the effect of STAT3 expression by Treg on tumorgrowth CD69 and IFN-γ expression. FIG. 46A shows that tumor growth, asassessed by tumor volume, progresses with delayed kinetic inSTAT3^(KO)Treg mice than it does in STAT3^(WT)Treg mice. FIGS. 46B-46C.show that STAT3^(KO)Treg mice have increased expression levels of CD69and IFN-γ compared to STAT3^(WT)Treg mice in CD8 TIL.

FIGS. 47A-47B show that anti-IL23-R antibody interacts with Tregs. FIG.47A shows that treatment of C57BL/6 mice with anti-IL-23R antibodyreduces tumor volumes compared to C57BL/6 mice treated with isotype.FIG. 47B shows that treatment of Rag2^(−/−) mice that have been injectedwith Tregs alone or in conjunction with CD8+ T cells with anti-IL-23Rantibody reduces tumor volume compared to treatment with isotype.

DETAILED DESCRIPTION OF INVENTION

The present disclosure, in one aspect, relates to the surprisingdiscovery that IL-23R modulates Treg differentiation, and in particular,that inhibition of IL-23R induces Treg differentiation. The IL-23Rcytokine receptor subunit dimerizes with IL-12 β1 to form the IL-23receptor, which binds the IL-23 cytokine. It has surprisingly been foundthat IL-23R is expressed on Treg cells the and inhibition of IL-23Rinduces differentiation of these Treg cells (e.g., to Teff cells).

The present disclosure, in another aspect, relates to the surprisingdiscovery that IL-23R modulates cancer. Regulatory T cells arecritically important for maintenance of a balanced immune system, inpart because they help to dampen excessive immune responses and preventautoimmunity. However, immune suppression exerted by Treg can impedeanti-tumor immune responses and promote cancer growth. Inducingdifferentiation of Treg via decreasing IL-23R activity has the capacityto slow cancer progression and agents that decrease IL-23R activityrepresent novel therapeutics for treating cancer.

Thus, in one aspect, the present disclosure provides agents for treatingcancer by decreasing IL-23R activity. In another aspect, the presentdisclosure provides agents for inducing differentiation of a regulatoryT (Treg) cell to an effector T cell by decreasing IL-23R activity. Suchagents can include antibodies, polypeptides, nucleic acids, or smallmolecules.

Interleukin-23 Receptor (IL-23R)

IL-23R mediates signaling by IL-23. Interleukin-23 (IL-23) is aheterodimeric cytokine comprised of two subunits, p19 which is unique toIL-23, and p40, which is shared with IL-12. IL-23 mediates signaling bybinding to the heterodimeric IL-23 receptor, comprised of an IL-23Rsubunit and an IL-12Rβ1, which is shared by the IL-12 receptor. The p19subunit of IL-23 interacts with the IL-23R subunit of the IL-23 receptorand the p40 subunit of IL-23 interacts with the IL-12Rβ1 subunit of theIL-23 receptor. IL-23 interacts with the IL-23 receptor at theextracellular region.

IL-23-R is a protein with isoforms ranging in size from 20 kDa to 43kDa. The sequence of IL-23-R can be found at NCBI Ref. No. NM_144701.2.

IL-23R associates with Janus kinase 2 (Jak2) and in a ligand-dependentmanner with Signal transducer and activator of transcription STAT3.IL-12Rβ1 interacts directly with Tyrosine kinase 2 (Tyk2).

IL-23 induced activation of STAT3 leads to direct binding ofphosphorylated STAT3 to Interleukin-17 (IL-17) and Interleukin 17F(IL-17F) promoters. STAT3 up-regulates the expression of Retinoic AcidReceptor-Related Orphan Receptor Gamma-T (ROR-gamma), a Th17 specifictranscriptional regulator that is critical for the expression of twomembers of Interleukin-17 family, IL-17A (IL-17) and IL-17F. IL-23induced JAK2 activation triggers Phosphoinositide-3-kinase(PI3K)/RAC-alpha serine/threonine kinase (AKT) and Nuclear factor kappaB(NF-kB) pathways which are required for IL-17 production. PI3K/AKTpathway is involved in STAT3 phosphorylation through an undeterminedmechanism.

In embodiments, an agent described herein decreases IL-23R activity. Ingeneral, a decrease means any statistically significant decrease inactivity by any one or more measures. The measures of activity may bedirect or indirect, using one or more of the measures described hereinor otherwise known to those of ordinary skill in the art. The measuremay be of a cell in a resting state or a cell contacted with exogenousligand to IL-23R. The measure may be as illustrated in the examples. Themeasure typically will be carried out in vitro, to establish that theagent has the desired properties of decreasing IL-23R activity but notIL-12R activity. Such agents then can be administered in therapeuticallyeffective amounts. Alternatively, the measure may be an improvement inthe subject being treated, such as the slowing of progression of, thehalting of the growth of, or the shrinkage of the cancer.

In some embodiments, IL-23R activity is decreased relative to a controlat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, and even more than 75%. In some embodiments, IL-23R activityis decreased 1%-5%, 1%-10%, 5%-10%, 5%-25%, 10%-50%, 20%-40%, 40%-60%,50%-90%, 50%-70%, 60%-80%, 80%-90%, 90%-95%, 90%-99%, or 95%-99%.

In some embodiments, an agent described herein is administered to asubject and IL-23R activity is decreased in the subject. In someembodiments, the agent is administered to a subject and IL-23R activityis decreased relative to the level of IL-23R activity prior toadministration of the agent. In some embodiments, the IL-23R activity isdecreased relative to the level of IL-23R activity in the subject 30minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours 12 hours 24hours, 2 days, 3 days, 4 days, 5 days 6 days, 1 week, 2 weeks, 3 weeks,or 4 weeks or more prior to administration of the agent. In someembodiments, multiple doses of the agent are administered to a subjectand IL-23R activity is decreased relative to the level of IL-23Ractivity prior to the first administration of the agent, e.g., thebeginning of treatment. In some embodiments, the IL-23R activity isdecreased relative to the level of IL-23R activity in the subject 30minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours 12 hours 24hours, 2 days, 3 days, 4 days, 5 days 6 days, 1 week, 2 weeks, 3 weeks,or 4 weeks or more prior to the first administration of the agent. Insome embodiments, multiple doses of the agent are administered to asubject and IL-23R activity is decreased relative to the level of IL-23Ractivity prior to the current administration of the agent. In someembodiments, the IL-23R activity is decreased relative to the level ofIL-23R activity in the subject 30 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours 12 hours 24 hours, 2 days, 3 days, 4 days, 5days 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks or more prior to thecurrent administration of the agent.

In some embodiments, the agent is administered to a subject and IL-23Ractivity is decreased relative to the level of IL-23R activity in acontrol subject who is not treated with the agent. In some embodiments,the control subject has the same or a similar type of cancer as thesubject. In some embodiments, the control subject has the same or asimilar severity of cancer as the subject. In some embodiments, thecontrol subject does not have cancer. In some embodiments, the controlsubject is the same or a similar age as the subject. In someembodiments, the control subject is the same sex as the subject. In someembodiments, the control subject is related to the subject. In someembodiments, the control subject is a twin of the subject.

In some embodiments, the agent is administered to a subject in amountseffective to treat cancer in the subject. Effective amounts aredescribed in greater detail below.

In some embodiments, Treg cells are contacted in vitro with an agentdescribed herein and IL-23R activity is decreased. In some embodiments,IL-23R activity is decreased relative a control cell, e.g., a celltreated with a compound known to not decrease IL-23R activity andIL-12Rβ1 activity. In some embodiments, IL-23R activity is decreasedrelative a cell of the same clonal population, e.g., of the sameculture, that are not administered the agent. In some embodiments, theIL-23R activity of the cell of the same clonal population is measuredsimultaneously or near simultaneously with the IL-23R activity of theTreg cell. In some embodiments, the IL-23R activity of the cell of thesame clonal population is measured before or after the IL-23R activityof the Treg cell. In some embodiments, the IL-23R activity is decreasedrelative to cells of the same cell type, e.g., are derived from the samesource, that are not administered the agent.

In some embodiments, a decrease in IL-23R activity comprises a decreasein expression of FoxP3, Helios, IL-17A, IL-17F, ROR-gamma, and/or IL-23.In some embodiments, expression of FoxP3, Helios, IL-17A, IL-17F,ROR-gamma, and/or IL-23 is decreased 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100%. In some embodiments, expression of FoxP3,Helios, IL-17A, IL-17F, ROR-gamma, and/or IL-23 is decreased more than1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%. In some embodiments, expression of FoxP3, Helios,IL-17A, IL-17F, ROR-gamma, and/or IL-23 is decreased 1%-5%, 1%-10%,5%-10%, 5%-25%, 10%-50%, 20%-40%, 40%-60%, 50%-90%, 50%-70%, 60%-80%,80%-90%, 90%-95%, 90%-99%, 90%-99%, or 95%-99%.

Gene expression can be measured at either the RNA or protein level.Assays for detecting RNA include, but are not limited to, Northern blotanalysis, RT-PCR, sequencing technology, RNA in situ hybridization(using e.g., DNA or RNA probes to hybridize RNA molecules present in thesample), in situ RT-PCR (e.g., as described in Nuovo G J, et al. Am JSurg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract.1994, 190: 1017-25), and oligonucleotide microarray (e.g., byhybridization of polynucleotide sequences derived from a sample tooligonucleotides attached to a solid surface (e.g., a glass wafer withaddressable location, such as Affymetrix microarray (Affymetrix®, SantaClara, CA)).

Assays for detecting protein levels include, but are not limited to,immunoassays (also referred to herein as immune-based or immuno-basedassays, e.g., Western blot, ELISA, proximity extension assays, andELISpot assays), Mass spectrometry, and multiplex bead-based assays.Other examples of protein detection and quantitation methods includemultiplexed immunoassays as described for example in U.S. Pat. Nos.6,939,720 and 8,148,171, and published U.S. Patent Application No.2008/0255766, and protein microarrays as described for example inpublished U.S. Patent Application No. 2009/0088329.

In some embodiments, IL-23 activity can be measured by measuring IL-17and/or interferon-gamma secretion following treatment of activatedpreipheral blood mononuclear cells (Dallas et al., Drug Metabolism andDisposition, 2013, 41:689-693). In some embodiments, IL-23 activity canbe measured by measuring differentiation of CD4+ T cells toIL-17-producing pathogenic Th17 cells. (Reddy et al., Cell Immunol,2007, 47:1-11).

In some embodiments, a decrease in IL-23R activity stimulates Teffdevelopment (i.e., Treg differentiation) as is described in furtherdetail below.

Interleuken 12

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 andp40 subunits. IL-12 plays a role in the differentiation of naïve T cellsinto T-helper type 1 CD4 lymphocytes that secrete IFNγ. IL-12 is alsoimportant for T cell dependent immune and inflammatory responses invivo. The IL-12 receptor (IL-12Rβ) is composed of IL-12Rβ1 and IL-12Rβ2subunits.

The main role of IL-12 is activation of interferon gamma production.Upon binding to its receptor, IL-12 activates Janus family kinasesTyrosine kinase 2 (Tyk2) and Janus kinase 2 (Jak2). Interleukin 12receptor, beta 1 (IL-12Rβ1) binds Tyk2, whereas Interleukin 12 receptor,beta 2 (IL-12Rβ2) associates with Jak2. Jak2 phosphorylates the tyrosineresidues of STAT3 and STAT4. They translocate to the nucleus and bind tothe promoter site of IFN-gamma. STAT4 also induces transcription ofIL-12Rβ2, IL18-Rβ1, Interferon regulatory factor 1 (IRF1), Interleukin 2receptor, alpha (IL-2R), and GCNT1 glucosaminyl (N-acetyl) transferase 1core 2 (G6NT).

In embodiments, agents described herein do not appreciably decreaseIL-12Rβ activity. It will be understood by one of ordinary skill in theart, that according to the invention, agents are employed that decreaseIL-23R activity and do not decrease IL-12Rβ activity. In general, it isexpected that such agents will not appreciably decrease IL-12Rβ activitybecause the agents do not interact directly with IL-12Rβ. The IL-12Rβactivity, therefore, will not vary more than would be expected incontrol cells.

In embodiments, the absence of a decrease in activity may be establishedby any one or more measures. The measures of activity may be direct orindirect, using one or more of the measures described herein orotherwise known to those of ordinary skill in the art. The measure maybe of a cell in a resting state or a cell contacted with exogenousligand to IL-12R. The measure may be as illustrated in the examples. Themeasure typically will be carried out in vitro, to establish that theagent has the desired properties of decreasing IL-23R activity but notIL-12Rβ activity. The measure typically will be carried out in vitro, toestablish that the agent has the desired property of not decreasingIL-12Rβ activity. As used herein “does not decrease” means IL-12Rβactivity will decrease significantly less than the decrease observed inIL-23 activity. that the IL-12Rβ activity levels do not decrease in astatistically significant manner, relative to a control. Typically, theIL-12Rβ activity does not decrease in a statistically significantmanner, relative to a control. In general, the decrease in IL-12Rβactivity, relative to a control, would be less than 25%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.4%, 0.2%, or 0.1%. Typically, there would be nostatistically significant decrease in IL-12Rβ activity.

In some embodiments, the agent is administered to a subject and IL-12Rβactivity is not decreased relative to the level of IL-12Rβ activityprior to administration of the agent. In some embodiments, the IL-12Rβactivity is not decreased relative to the level of IL-12Rβ activity inthe subject 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours 12 hours 24 hours, 2 days, 3 days, 4 days, 5 days 6 days, 1 week,2 weeks, 3 weeks, or 4 weeks or more prior to administration of theagent. In some embodiments, multiple doses of the agent are administeredto a subject and IL-12Rβ activity is not decreased relative to the levelof IL-12Rβ activity prior to the first administration of the agent,e.g., the beginning of treatment. In some embodiments, the IL-12Rβactivity is not decreased relative to the level of IL-12Rβ activity inthe subject 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours 12 hours 24 hours, 2 days, 3 days, 4 days, 5 days 6 days, 1 week,2 weeks, 3 weeks, or 4 weeks or more prior to the first administrationof the agent. In some embodiments, multiple doses of the agent areadministered to a subject and IL-12Rβ activity is not decreased relativeto the level of IL-12Rβ activity prior to the current administration ofthe agent. In some embodiments, the IL-12Rβ activity is not decreasedrelative to the level of IL-12Rβ activity in the subject 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours 12 hours, 24 hours, 2days, 3 days, 4 days, 5 days 6 days, 1 week, 2 weeks, 3 weeks, or 4weeks or more prior to the current administration of the agent.

In some embodiments, the agent is administered to a subject and IL-12Rβactivity is not decreased relative to the level of IL-12Rβ activity in acontrol subject who is not treated with the agent. In some embodiments,the control subject has the same or a similar type of cancer as thesubject. In some embodiments, the control subject has the same or asimilar severity of cancer as the subject. In some embodiments, thecontrol subject does not have cancer. In some embodiments, the controlsubject is the same or a similar age as the subject. In someembodiments, the control subject is the same sex as the subject. In someembodiments, the control subject is related to the subject. In someembodiments, the control subject is a twin of the subject.

In some embodiments, the agent is administered to a Treg cell in vitroand IL-12Rβ activity is not decreased relative a cell of the same clonalpopulation, e.g., of the same culture, that are not administered theagent. In some embodiments, the IL-12Rβ activity of the cell of the sameclonal population is measured simultaneously or near simultaneously withthe IL-12Rβ activity of the Treg cell. In some embodiments, the IL-12Rβactivity of the cell of the same clonal population is measured before orafter the IL-12Rβ activity of the Treg cell. In some embodiments, theagent is administered to a Treg in vitro and IL-12Rβ activity is notdecreased relative to cells of the same cell type, e.g., are derivedfrom the same source, that are not administered the agent.

In some embodiments, IL-12Rβ activity comprises STAT4 phosphorylation.In some embodiments, STAT4 phosphorylation levels do not decrease bymore than 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

In some embodiments, IL-12Rβ activity comprises a change in expressionof IL-12Rβ2, IL18-12Rβ1, IRF1, IL-2R, and/or G6NT. In some embodiments,expression of IL-12Rβ2, IL18-12Rβ1, IRF1, IL-2R, and/or G6NT do notdecrease by more than 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

In some embodiments, IL12 activity can be measured by measuring IL-17and/or interferon-gamma secretion following treatment of activatedperipheral blood mononuclear cells (Dallas et al., Drug Metabolism andDisposition, 2013, 41:689-693). In some embodiments, IL-23 activity canbe measured by measuring differentiation of CD4+ T cells tointerferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells. (Reddy etal., Cell Immunol, 2007, 47:1-11.)

Induction of Treg Differentiation

In some embodiments, the agents described herein are useful in a methodof inducing differentiation of cells. In some embodiments, an agentdescribed herein induces differentiation of a T cell. In someembodiments, an agent described herein induces differentiation of aregulatory T cell (i.e., a Treg). In some embodiments, an agentdescribed herein induces differentiation of a Treg cell to a Teff cell.

In some embodiments, Treg cells are Helios+ Treg cells. In someembodiments, Treg cells are TIGIT+ Treg cells. In some embodiments, Tregcells are CD4⁺ Treg cells. In some embodiments, the CD4+ Treg cells areCD4+CD25+ Treg cells. CD4+CD25+ regulatory T cells are thought tofunction as a regulator of autoimmunity by suppressing the proliferationand/or cytokine production of CD4+CD25-T cell responder cells at thesite of inflammation. In some embodiments, the CD4+ Treg cells areCD4+FoxP3+ Treg cells. In some embodiments, the CD4+ Treg cells areCD4+FoxP3+CD25+ Treg cells.

In some embodiments, Treg cells are CD8⁺ Treg cells. In someembodiments, the CD8⁺ Treg cells are CD8+CD28− Treg cells. In someembodiments, the CD8+ Treg is positive for Killer cell immunoglobulinlike receptor (Kir+).

In some embodiments, Treg cells are Tr1 Treg cells. In some embodiments,Treg cells are Th3 Treg cells. In some embodiments, Treg cells are Qa-1restricted Treg cells.

In some embodiments, Teff cells are marked by the expression of certaincytokines. Examples of effector cytokines expressed by Teff includetumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ),interleukin-17 (IL-17), interleukin-2 (IL-2), and Granzyme B. However,the skilled artisan appreciates that other effector cytokines may alsobe expressed by differentiated T effector cells.

In some embodiments, effector cells are CD4⁺ effector T cells. As usedherein the term “CD4 effector T cells” refers to a subset of T cellswhich are associated with cell-mediated immune response. They arecharacterized by the secretion of one or more effector cytokines suchas, but not limited to, IFN-γ, TNF-α, IL-17, IL-2 and granzyme B.

In some embodiments, the effector cells are CD8⁺ effector T cells. Insome embodiments, CD8⁺ effector T cells are cytotoxic T cells.

In some embodiments, when differentiation is induced, 0.01%-100% of theTreg, e.g., intratumoral Treg, are converted to Teff. In someembodiments, when differentiation is induced, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%,8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of the Treg, e.g., intratumoral Treg, are converted to Teff. Insome embodiments, when differentiation is induced, more than 0.01%,0.05%, 0.1%, 0.5%, 1%, 5%, 10%, or 20% of the Treg, e.g., intratumoralTreg, are converted to Teff. In some embodiments, when differentiationis induced, 0.01%-1%, 0.1%-1%, 0.5%-2%, 1%-10%, 2%-15%, 5%-20%, 10%-30%,20%-50%, 20%-70%, 40%-50%, 40%-60%, 50%-70%, 50%-80%, 60%-70%, 60%-90%,70%-90%, 70%-99%, or 80%-99% of the Treg, e.g., intratumoral Treg, areconverted to Teff.

In some embodiments, when differentiation is induced, 0.01%-100% ofCD4+CD2530 cells, e.g., intratumoral CD4+CD25+ cells, are converted toCD8+ cells. In some embodiments, when differentiation is induced, 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%,5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% of CD4+CD25+ cells, e.g., intratumoralCD4+CD25+ cells, are converted to CD8+ cells. In some embodiments, whendifferentiation is induced, more than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%,10%, or 20% of CD4+CD25+ cells, e.g., intratumoral CD4+CD25+ cells, areconverted to CD8+ cells. In some embodiments, when differentiation isinduced, 0.01%-1%, 0.1%-1%, 0.5%-2%, 1%-10%, 2%-15%, 5%-20%, 10%-30%,20%-50%, 20%-70%, 40%-50%, 40%-60%, 50%-70%, 50%-80%, 60%-70%, 60%-90%,70%-90%, 70%-99%, or 80%-99% of CD4+CD25+ cells, e.g., intratumoralCD4+CD25+ cells, are converted to CD8+ cells.

In some embodiments, when differentiation is induced, 0.01%-100% ofCD4+CD25+ cells, e.g., intratumoral CD4+CD25+ cells, are converted tocells that secrete one or more of IFN-γ, TNF-α, IL-17, IL-2 and granzymeB. In some embodiments, when differentiation is induced, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%,6.0%, 7.0%, 8.0%, 9.0%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% of CD4+CD25+ cells, e.g., intratumoral CD4+CD25+cells, are converted to cells that secrete one or more of IFN-γ, TNF-60, IL-17, IL-2 and granzyme B. In some embodiments, when differentiationis induced, more than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, or 20% ofCD4+CD25+ cells, e.g., intratumoral CD4+CD25+ cells, are converted tocells that secrete one or more of IFN-γ, TNF-α, IL-17, IL-2 and granzymeB. In some embodiments, when differentiation is induced, 0.01%-1%,0.1%-1%, 0.5%-2%, 1%-10%, 2%-15%, 5%-20%, 10%-30%, 20%-50%, 20%-70%,40%-50%, 40%-60%, 50%-70%, 50%-80%, 60%-70%, 60%-90%, 70%-90%, 70%-99%,or 80%-99% of CD4+CD25+ cells, e.g., intratumoral CD4+CD25+cells, areconverted to cells that secrete one or more of IFN-γ, TNF-α, IL-17, IL-2and granzyme B.

In some embodiments, the phenotype of the cells is determined using anymethod known in the art, for example, flow cytometry.

In some embodiments, the cells are differentiated in vitro. In someembodiments, In some embodiments, one or more parameters identifiedherein, e.g., IL-23R or IL-12Rβ activity, stat3 phosphorylation,expression of one or more targets of IL-23R and/or IL-12Rβ, and/orconversion of Treg to Teff, are measured in the in vitro cells.

Agents

The methods described herein contemplate treating cancer with agentsthat decrease IL-23R activity and do not decrease IL-12Rβ activity.

Examples of inhibitor agents that may be used in accordance with thepresent disclosure include, without limitation, nucleic acids (e.g., DNAand/or RNA) and nucleic acid analogues; antibodies, includingfull-length antibodies and antigen-binding antibody fragments,polyclonal antibodies, monoclonal antibodies, chimeric antibodies, andhumanized antibodies; proteins, including peptides, peptide-mimetics andaptamers; and small molecules.

In some embodiments, an agent described herein binds the extracellulardomain of IL-23R.

Antibodies

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (i.e., full-length) polyclonal or monoclonal antibodies,but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)₂,Fv), single chain (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. An antibody includes an antibody of any class, suchas IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibodyneed not be of any particular class. Depending on the antibody aminoacid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The antibodies to be used in the methods described herein can be murine,rat, human, or any other origin (including chimeric or humanizedantibodies). In some examples, the antibody comprises a modifiedconstant region, such as a constant region that is immunologicallyinert, e.g., does not trigger complement mediated lysis, or does notstimulate antibody-dependent cell mediated cytotoxicity (ADCC). ADCCactivity can be assessed using methods disclosed in U.S. Pat. No.5,500,362. In other embodiments, the constant region is modified asdescribed in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No.PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.

Any of the antibodies described herein can be either monoclonal orpolyclonal. A “monoclonal antibody” refers to a homogenous antibodypopulation and a “polyclonal antibody” refers to a heterogenous antibodypopulation. These two terms do not limit the source of an antibody orthe manner in which it is made.

In one example, the antibody used in the methods described herein is ahumanized antibody. Humanized antibodies refer to forms of non-human(e.g., murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or antigen-binding fragments thereof that containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs “derived from” one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

In another example, the antibody described herein is a chimericantibody, which can include a heavy constant region and a light constantregion from a human antibody. Chimeric antibodies refer to antibodieshaving a variable region or part of variable region from a first speciesand a constant region from a second species. Typically, in thesechimeric antibodies, the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals (e.g., a non-human mammal such as mouse, rabbit, and rat), whilethe constant portions are homologous to the sequences in antibodiesderived from another mammal such as human. In some embodiments, aminoacid modifications can be made in the variable region and/or theconstant region.

In some examples, the antibody disclosed herein specifically binds atarget antigen, such as IL-23R. In some embodiments, the antibodydisclosed herein specifically binds the extracellular domain of IL-23R.An antibody that “specifically binds” (used interchangeably herein) to atarget or an epitope is a term well understood in the art, and methodsto determine such specific binding are also well known in the art. Amolecule is said to exhibit “specific binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular target antigen than it does withalternative targets. An antibody “specifically binds” to a targetantigen if it binds with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other substances. For example, anantibody that specifically (or preferentially) binds to an IL-23Repitope is an antibody that binds this IL-23R epitope with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other IL-23R epitopes or non-IL-23R epitopes. It is alsounderstood by reading this definition that, for example, an antibodythat specifically binds to a first target antigen may or may notspecifically or preferentially bind to a second target antigen. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

Antibodies capable of interfering with the IL-23R signaling pathway canbe an antibody that binds an IL-23R (e.g., the extracellular domain ofIL-23R) and inhibits IL-23R biological activity and/or downstreampathways mediated by IL-23R. Exemplary antibodies that bind IL-23R areshown in Table 1. In some embodiments, the antibodies shown in Table 1bind the extracellular domain of IL-23R.

TABLE 1 Anti-IL23-R Antibodies Host Species Source Catalog No. SpeciesReactivity Clonality Immunogen Conjugated ? Invitrogen MA-24804 MouseHuman, Monoclonal Amino acids No mouse, 288-315 of non-human human IL-primate 23R Invitrogen MA5-23906 Rat Mouse Monoclonal Recombinant Nomouse IL-23 R Gly24- Asp372 LifeSpan LS-C83986 Goat Human PolyclonalAmino acids No Biosciences 59-87 of human IL- 23R LifeSpan LS-C37068-100Mouse Human Monoclonal NP_653302 No Biosciences LifeSpan LS-C37069-100Mouse Human Monoclonal Unknown PE Biosciences LifeSpan LS-C125821-100Mouse Human Monoclonal Unknown PerCP Biosciences LifeSpan ABIN393707Mouse Human Monoclonal Unknown No Biosciences Aviva OABF00713 RabbitHuman, Polyclonal KLH- No Systems mouse, rat conjugated Biologysynthetic peptide of human IL- 23R United 223595 Rabbit Human PolyclonalFull-length No States human IL- Biological 23R Abeomics 10-4030 MouseHuman Monoclonal 250-450 of No IL-23R St. John's STJ24181 Rabbit Human,Polyclonal Recombinant No Laboratory Mouse, Rat peptide from humanIL-23R Biorbyt Orb140302 Rabbit Human Polyclonal Amino acids No 428-457of human IL- 23R Biorbyt Orb47095 Rabbit Human Polyclonal Full length Nohuman IL- 23R ProSci, Inc. 58-876 Rabbit Human Polyclonal Amino acids No428-457 of human IL- 23R Atlas HPA056427 Rabbit Human Polyclonal Fulllength No Antibodies human IL- 23R MyBioSource MBS854115 Rabbit HumanPolyclonal Full length No human IL- 23R MyBioSource MBS9404638 RabbitHuman, Polyclonal Full length No Mouse, Rat human IL- 23R ABclonal A1613Rabbit Human, Polyclonal Recombinant No Mouse, Rat Human ProteinInvitrogen MA5-23935 Mouse Human Monoclonal Mouse- No myeloma cell lineNSO-derived recombinant IL-23R amino acids 24-354 Invitrogen MA5-23906Rat Mouse Monoclonal Mouse- No myeloma cell line NSO-derived recombinantIL-23R amino acids 24-372 BioMatik CAC07795 Rabbit Human, PolyclonalHuman IL- No Mouse 23R amino acids 15-125 BioMatik CAU22706 Rabbit RatPolyclonal IL-23R No amino acids 25-355 Origene TA327101 Rabbit Human,Polyclonal Recombinant No Technologies Mouse, Rat human IL- 23R OrigeneTA321935 Rabbit Human, Polyclonal Human IL- No Technologies Mouse, Rat23R amino acids 21-227 LifeSpan LS-C403797 Rabbit Human Polyclonal HumanIL- No Biosciences 23R LifeSpan LS-C192978 Rabbit Human, PolyclonalHuman IL- No Biosciences Mouse, Rat 23R Abnova PAB0224 Rabbit Human,Polyclonal Human IL- No Mouse 23R amino acids 400- 470 Abnova H00149233Mouse Human Monoclonal Human IL- No 23R amino acids 553- 628 NSJ R32848Rabbit Mouse, Rat Polyclonal Amino acids No Bioreagents 25-233Antibodies ABIN342781 Goat Human Polyclonal Human IL- No Online 23Ramino acids 59-87 Antibodies ABIN221348 Mouse Human Monoclonal Human IL-PE Online 23R amino (Phycoery- acids 24-354 thrin) Antibodies ABIN530970Mouse Human Monoclonal LNQGECSS No Online PDIQNSVE EETTMLLE NDSPSETIPEQTLLPDE FVSCLGIVN EELPSINTY FPQNILESH FNRISLLE Antibodies ABIN360919Mouse Human Monoclonal Recombinant No Online protein of IL- 23Rextracellular domain Signalway 31023 Rabbit Human Polyclonal Human IL-No Antibody 23R amino acids 31-227 Signalway 32341 Rabbit Human,Polyclonal Human IL- No Antibody Mouse, Rat 23R United 036998 RabbitHuman Polyclonal Human IL- No States 23R amino Biological acids 427- 457United 128452 Mouse Human Monoclonal 553-629 No States from humanBiological IL23R United 247572 Mouse Unknown Monoclonal NP_653302, NoStates 553aa-628aa Biological United 247573 Mouse Unknown MonoclonalIL23R No States Biological Bioworld BS6809 Rabbit Human, PolyclonalHuman IL- No Technology Mouse, Rat 23R full- length protein AbbexaAbx130103 Rabbit Rat Polyclonal IL-23R No Cloud Clone MAE765Hu21 MouseHuman Monoclonal Human IL- No 23R Cloud Clone PAE765Hu21 Rabbit HumanPolyclonal Human IL- No 23R Enzo Life ALX-210- Goat Human, PolyclonalHuman IL- No Sciences 636-C200 Mouse 23R amino acids 59-87 Bio-RadVMA00297KT Mouse Human Monoclonal Human IL- No 23R R&D FAB14001P MouseHuman Monoclonal Human IL- PE Systems 23R amino (Phycoery- acids 24-354thrin) R&D FAB14001A Mouse Human Monoclonal Human IL- Allophyco- Systems23R amino cyanin acids 24-354 Bio-Rad AHP1821 Goat Mouse, PolyclonalHuman IL- No Human 23R N- terminus Santa Cruz sc-293485 Mouse HumanMonoclonal IL-23R No Biotechnology, Inc. Bio-Rad VMA00297K Mouse HumanMonoclonal IL-23 R No Novus NB600-1147 Rabbit Human, Polyclonal HumanIL- No Biologicals Mouse 23R amino acids 439- 455 Novus NB2-47419 RabbitHuman Polyclonal Human IL- No Biologicals 23R amino acids 258- 346 NovusNBP2- Mouse Human, Monoclonal Amino acids No Biologicals 27091SS Primate288-315 of hIL-23 receptor Bioss Bs-1460R Rabbit Human, Polyclonal HumanIL- No Mouse, Rat 23R BosterBio A00607 Rabbit Mouse, Rat PolyclonalMouse IL- No 23R amino acids 25-233 Millipore 06-1331 Rabbit Human,Polyclonal Human IL- No Sigma Mouse, 23R Rat, Ox, Horse Sigma-WH01429233M1 Mouse Human Monoclonal Human IL- No Aldrich 23R CreativeTAB-421CL Unknown Human Monoclonal Human IL- Unknown Biolabs 23RBiolegend Unknown: Clone Rat Mouse Monoclonal Mouse IL- No 12B2B64 23Ramino acids 24-374 R&D Unknown: Clone Unknown Mouse Monoclonal UnknownUnknown 258018

In some embodiments, the antibody has one, two or all of a light chainCDR1, CDR2, and/or CDR3 and/or one, two or all of a heavy chain CDR1,CDR2 and/or CDR3. In some embodiments, the antibody has an amino acidsequence having one, two or all of a light chain CDR1, CDR2, and/or CDR3and/or one, two or all of a heavy chain CDR1, CDR2 and/or CDR3 of anantibody in Table 1. In some embodiments, the antibody has an amino acidsequence having at least one, two or three modifications (e.g.,substitutions, e.g., conservative substitutions) but not more than 30,20 or 10 modifications (e.g., substitutions, e.g., conservativesubstitutions) to one, two or all of a light chain CDR1, CDR2, and/orCDR3 and/or one, two or all of a heavy chain CDR1, CDR2 and/or CDR3 ofan antibody in Table 1.

In some embodiments, the antibody has one or both of a VH and VL. Insome embodiments, the antibody has an amino acid sequence having one orboth of a VH and VL of an antibody in Table 1, or a sequence with 95-99%identity thereof, e.g., 95%, 96%, 97%, 98% or 99% identity, e.g.,96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity.

In some embodiments, the antibody binds IL-23R and does not bindIL-12Rβ. In some embodiments, the IL-12Rβ binding affinity of ananti-IL23R antibody measured. In some embodiments, the anti-IL23-Rantibodies shown in Table 1 are screened to identify anti-IL23-Rantibodies with low or no binding affinity for IL-12Rβ, e.g., hasgreater binding affinity for IL-23R than for IL-12Rβ. Binding affinitycan be expressed KD or dissociation constant, and an increased bindingaffinity corresponds to a decreased KD. One way of determining bindingaffinity of antibodies to IL-23R is by measuring binding affinity ofmonofunctional Fab fragments of the antibody. To obtain monofunctionalFab fragments, an antibody (for example, IgG) can be cleaved with papainor expressed recombinantly. The affinity of an anti- IL-23R Fab fragmentof an antibody can be determined by surface plasmon resonance(BIAcore3000™ surface plasmon resonance (SPR) system, BIAcore, INC,Piscaway N.J.). Kinetic association rates (kon) and dissociation rates(koff) (generally measured at 25° C.) are obtained; and equilibriumdissociation constant (KD) values are calculated as koff/kon. In someembodiments, an antibody described herein has 1, 2, 5, 10, 20, 50, 75,100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 5,000,10,000, 50,000, 100,000-fold or more greater binding affinity for IL-23Rthan for IL-12Rβ. In some embodiments, an antibody described herein hasgreater than 1, 2, 5, 10, 20, 50, 75, 100, 200, 300, 400, 500, 600, 700,800, 900, 1,000, 2,000, 5,000, 10,000, 50,000, 100,000-fold more bindingaffinity for IL-23R than for IL-12Rβ.

Methods of producing antibodies (e.g., monoclonal antibodies orpolyclonal antibodies) are known in the art. For example, a polyclonalantibody may be prepared by immunizing an animal, preferably a mammal,with an allergen of choice followed by the isolation ofantibody-producing B-lymphocytes from blood, bone marrow, lymph nodes,or spleen. Alternatively, antibody-producing cells may be isolated froman animal and exposed to an allergen in vitro against which antibodiesare to be raised. The antibody-producing cells may then be cultured toobtain a population of antibody-producing cells, optionally after fusionto an immortalized cell line such as a myeloma. In some embodiments, asa starting material B-lymphocytes may be isolated from the tissue of anallergic patient, in order to generate fully human polyclonalantibodies. Antibodies may be produced in mice, rats, pigs (swine),sheep, bovine material, or other animals transgenic for the humanimmunoglobulin genes, as starting material in order to generate fullyhuman polyclonal antibodies. In some embodiments, mice or other animalstransgenic for the human immunoglobulin genes (e.g. as disclosed in U.S.Pat. No. 5,939,598), the animals may be immunized to stimulate the invivo generation of specific antibodies and antibody producing cellsbefore preparation of the polyclonal antibodies from the animal byextraction of B lymphocytes or purification of polyclonal serum.

Monoclonal antibodies are typically made by cell culture that involvesfusing myeloma cells with mouse spleen cells immunized with the desiredantigen (i.e., hyrbidoma technology). The mixture of cells is dilutedand clones are grown from single parent cells on microtitre wells. Theantibodies secreted by the different clones are then assayed for theirability to bind to the antigen (with a test such as ELISA or AntigenMicroarray Assay) or immuno-dot blot. The most productive and stableclone is then selected for future use.

In some embodiments, the antibodies described herein are “humanized” foruse in human (e.g., as therapeutics). In some embodiments, an antibodyshown in Table 1 is a murine antibody and the antibody is humanized.“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. Humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region of the recipientare replaced by residues from a hypervariable region of a non-humanspecies (donor antibody) such as mouse, rat, rabbit or non-human primatehaving the desired antibody specificity, affinity, and capability. Insome instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

Nucleic Acids

In some embodiments, the agent is a nucleic acid. In some embodiments,the agent binds a nucleic acid expressing IL-23R. e.g., having asequence shown at NCBI Ref. No. NM_144701.2. In some embodiments, theagent binds a sequence encoding the extracellular domain of IL-23R.

As used herein, “gene silencing,” refers to post-transcriptional genesilencing, which may be the result of mRNA of a particular gene beingdegraded or blocked. The RNAi inhibitor agents provide herein may, insome embodiments, decrease the expression level of IL-23R mRNA by atleast about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99% or about 100% of the mRNA level found in thecell without the presence of an RNAi inhibitor agent.

As used herein, “RNA interference” is a biological process in which RNAmolecules inhibit gene expression, typically by causing the degradationof specific mRNA molecules.

In some embodiments, the agent is an siRNA. As used herein, “siRNA” is aclass of double-stranded RNA molecules, which interferes with theexpression of specific genes having a nucleotide sequence complementaryto the siRNA. siRNAs typically have a well-defined structure: a short(e.g., 21 base pair) double-stranded RNA (dsRNA) with phosphorylated 5′ends and hydroxylated 3′ ends with two overhanging nucleotides. TheDicer enzyme catalyzes production of siRNAs from long dsRNAs and smallhairpin RNAs (shRNAs). An siRNA for use in accordance with the presentdisclosure may be about 15 to about 35 base pairs, or about 20 to about25 base pairs, in length. In some embodiments, the siRNA may be about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34 or 35 base pairs in length. In some embodiments, the siRNA iscomplementary to IL-23R, e.g., having a sequence shown at NCBI Ref. No.NM_144701.2. In some embodiments, the siRNA is complementary to asequence encoding the extracellular domain of IL-23R.

Exemplary siRNAs against IL23-R are shown in Table 2. In someembodiments, the siRNA comprises a nucleic acid sequence of any one ofSEQ ID NOs; 19-31. In some embodiments, the siRNA comprises a nucleicacid sequence having 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NOs; 19-31.In some embodiments, the siRNA comprises a nucleic acid sequence havingat least 5, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or 21 consecutive nucleotides of a nucleic acid sequence of any oneof SEQ ID NOs; 19-31, or a sequence having 70%, 71%. 72%. 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to anucleic acid sequence having at least 5, e.g., 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of anucleic acid sequence of any one of SEQ ID NOs; 19-31.

TABLE 2 siRNAs against IL23-R generated using siRNA Wizard v3.1 athttp://www.invivogen.com/sirnawizard/design.php Number Sequence PositionLength (bp) SEQ ID NO:  1 GACCTGGAACGTGAAAGAATT   27-48 21 19  2GCAAAGCGTTTCAGCATGATA  209-230 21 20  3 GATTCCGAAATGGCTGTATGA  411-43221 21  4 GAAATGGCTGTATGAAGATAT  417-438 21 22  5 GCGAACTGATGAACAACAACA 485-506 21 23  6 GGTGTATATTCCGGATCTGAA  660-681 21 24  7GGATCTGAACACCGGCTATAA  672-693 21 25  8 GCAGCCATCTGAGCAACAACA  673-69421 26  9 GCAGCCATCTGAGCAACAACA  722-743 21 27 10 GCAACAACAACGAAATTACCA 734-755 21 28 11 GCGAACTGAGCCTGATTCTGA  878-899 21 29 12GGGCATTGTGAACGAAGAACT 1032-1053 21 30 13 GCCGAGCATTAACACCTATTT 1053-107421 31

In some embodiments, the agent is an shRNA. As used herein, “shRNA”refers to a sequence of RNA that makes a tight hairpin turn that can beused to silence target gene expression through RNA interference (RNAi).Expression of shRNA in cells may be accomplished by delivery of plasmidsor through viral or bacterial vectors. For example, in some embodiments,shRNA targeting IL-23R may be delivered to Treg cells by transfectingthe cells with a plasmid that contains a nucleic acid encoding theshRNA. In some embodiments, bacterial vectors may be used to obtainshRNA expression in cells. In some embodiments, viral vectors (e.g.,adeno-associated viruses (AAVs), adenoviruses, and lentiviruses) may beused to obtain shRNA expression in cells. Due to the ability of shRNA toprovide specific, long-lasting, gene silencing, shRNA may be used forgene therapy applications. In some embodiments, the shRNA iscomplementary to IL-23R, e.g., having a sequence shown at NCBI Ref. No.NM_144701.2. In some embodiments, the shRNA is complementary to asequence encoding the extracellular domain of IL-23R.

As used herein, a “stem-loop structure” refers to a nucleic acid havinga secondary structure that includes a region of nucleotides which areknown or predicted to form a double strand (stem portion) that is linkedon one side by a region of predominantly single-stranded nucleotides(loop portion). The term “hairpin” may be used herein to refer to astem-loop structure.

Peptides

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof.

A peptide that is “derived from” a protein (e.g., a peptide derived fromIL-23R) means the peptide is obtained from the protein and has an aminoacid sequence that shares homology with the fragment of the protein itcorresponds to. The amino acid sequence of the peptide may be at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99% or 100% identical to the amino acid sequence of thefragment of the protein it corresponds to. A peptide that is derivedfrom a protein may also contain chemical modifications, amino acidsubstitutions, and/or unnatural amino acids.

Useful peptides ing to the invention include antibodies, zinc fingerproteins which can target and cleave DNA, and protein antagonists whichmimic the structure of the native ligand for a receptor and blockbinding of the native ligand without agonizing the receptor.

Small Molecules

In other embodiments, the IL-23R inhibitory compounds described hereinare small molecules, which can have a molecular weight of about any of100 to 20,000 daltons, 500 to 15,000 daltons, or 1000 to 10,000 daltons.Libraries of small molecules are commercially available. The smallmolecules can be administered using any means known in the art,including inhalation, intraperitoneally, intravenously, intramuscularly,subcutaneously, intrathecally, intraventricularly, orally, enterally,parenterally, intranasally, or dermally. In general, when the agentaccording to the invention is a small molecule, it will be administeredat the rate of 0.1 to 300 mg/kg of the weight of the patient dividedinto one to three or more doses. For an adult patient of normal weight,doses ranging from 1 mg to 5 g per dose can be administered.

The above-mentioned small molecules can be obtained from compoundlibraries. The libraries can be spatially addressable parallel solidphase or solution phase libraries. See, e.g., Zuckermann et al. J. Med.Chem. 37, 2678-2685, 1994; and Lam Anticancer Drug Des. 12:145, 1997.Methods for the synthesis of compound libraries are well known in theart, e.g., DeWitt et al. PNAS USA 90:6909, 1993; Erb et al. PNAS USA91:11422, 1994; Zuckermann et al. J. Med. Chem. 37:2678, 1994; Cho etal. Science 261:1303, 1993; Carrell et al. Angew Chem. Int. Ed. Engl.33:2059, 1994; Carell et al. Angew Chem. Int. Ed. Engl. 33:2061, 1994;and Gallop et al. J. Med. Chem. 37:1233, 1994. Libraries of compoundsmay be presented in solution (e.g., Houghten Biotechniques 13:412-421,1992), or on beads (Lam Nature 354:82-84, 1991), chips (Fodor Nature364:555-556, 1993), bacteria (U.S. Pat. No. 5,223,409), spores (U.S.Pat. No. 5,223,409), plasmids (Cull et al. PNAS USA 89:1865-1869, 1992),or phages (Scott and Smith Science 249:386-390, 1990; Devlin Science249:404-406, 1990; Cwirla et al. PNAS USA 87:6378-6382, 1990; Felici J.Mol. Biol. 222:301-310, 1991; and U.S. Pat. No. 5,223,409).

In Vitro Differentiation

In some embodiments, cells, e.g., Treg cells, are contacted in vitrowith an agent described herein and IL-23R activity is decreased. In someembodiments, differentiation is induced.

In some embodiments, the cells, e.g., Treg cells, are isolated. In someembodiments, the cells are isolated from mice. In some embodiments, thecells are isolated from murine spleens. In some embodiments, the cellsare isolated from murine lymph nodes. In some embodiments, the cells,e.g., Treg cells, are isolated from a human subject. In someembodiments, the cells are isolated from peripheral blood mononuclearcells.

In some embodiments, the cells, e.g., Treg cells, are contacted in abuffer, e.g., PBS. In some embodiments, the Treg cells are contacted ina cell culture media, e.g., a hematopietic cell culture medium (e.g.,X-VIVO®15) or a T cell expansion media (e.g., CTS OpTmizer T CellExpansion SFM®).

In some embodiments, the cells, e.g., Treg cells, are contacted in thepresence of anti-CD3 antibodies. In some embodiments, the cells, e.g.,Treg cells are contacted in the presence of anti-CD28 antibodies. Insome embodiments, the cells, e.g., Treg cells are contacted in thepresence of anti-CD3 antibodies and anti-CD28 antibodies. In someembodiments, the anti-CD3 antibodies and/or anti-CD28 antibodies areattached to a surface. In some embodiments, the anti-CD3 antibodiesand/or anti-CD28 antibodies are attached to a plate. In someembodiments, the anti-CD3 antibodies and/or anti-CD28 antibodies areattached to a bead.

In some embodiments, the cells, e.g., Treg cells, are contacted in thepresence of one or more inflammatory cytokines. Inflammatory cytokinesinclude, for example, interleukin-1 (IL-1), tumor necrosis factor (TNF),gamma-interferon (IFN-gamma), IL-12, IL-18, granulocyte-macrophagecolony stimulating factor, IL-2, IL-4, IL-10, IL-13, IFN-alpha andTGF-β.

In some embodiments, the cells, e.g., Treg cells, are contacted in thepresence of IL-2. In some embodiments, T reg cells are contacted in0-1000 ng, 0-500 ng, 0-200 ng, 0-100 ng, or 0-50 ng IL-2. In someembodiments, the cells, e.g., Treg cells, are contacted in the presenceof IL-4. In some embodiments, the T reg cells are contacted in 0-1000ng, 0-500 ng, 0-200 ng, 0-100 ng, 0-50 ng, or 0-20 ng IL-4.

Therapeutic Uses

In some embodiments, provided herein are methods for treating cancer byadministering an agent described herein.

The subject to be treated by the methods described herein are human.

A human subject who needs the treatment may be a human patient having,at risk for, or suspected of having cancer. A subject having cancer canbe identified by routine medical examination, e.g., laboratory tests,functional tests, biopsy, CT scans, or ultrasounds. A subject suspectedof having cancer might show one or more symptoms of the disorder. Asubject at risk for cancer can be a subject having one or more of therisk factors for that disorder. For example, risk factors associatedwith cancer include (a) hereditary cancer, (b) age, and (c) familyhistory of cancer.

Cancers include but are not limited to: Oral: buccal cavity, lip,tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: non-small cell lung cancer (NSCLC), small cell lungcancer, bronchogenic carcinoma (squamous cell or epidermoid,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, larynx,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or smallintestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi'ssarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), largebowel or large intestines (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma), rectal, colon, colon-rectum, colorectal;Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), head and neck cancer, meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastomamultiform, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord neurofibroma, meningioma, glioma, sarcoma);Gynecological: uterus (endometrial carcinoma), cervix (cervicalcarcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma[serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, SertolI-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia [acute and chronic], acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma; medullary thyroid carcinoma, multiple endocrine neoplasiatype 2A, multiple endocrine neoplasia type 2B, familial medullarythyroid cancer, pheochromocytoma, paraganglioma; and Adrenal glands:neuroblastoma.

“An effective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size, gender and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used, that is, the highest safedose according to sound medical judgment. It will be understood by thoseof ordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of cancer. Alternatively, sustained continuous releaseformulations of agent that decreases IL-23R activity may be appropriate.Various formulations and devices for achieving sustained release areknown in the art.

In one example, dosages for an agent that decreases IL-23R activity asdescribed herein may be determined empirically in individuals who havebeen given one or more administration(s) of the agent. Individuals aregiven incremental dosages of the agent. To assess efficacy of the agent,an indicator of IL-23R activity can be followed.

For the purpose of the present disclosure, the appropriate dosage of anagent that decreases IL-23R activity will depend on the specific agents(or compositions thereof) employed, the type and severity of cancer,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has cancer, a symptom of cancer, or a predisposition towardthe disease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect the disorder, the symptom of thedisease, or the predisposition toward the disease.

Alleviating cancer includes delaying the development or progression ofthe disease, or reducing disease severity. Alleviating the disease doesnot necessarily require curative results. As used therein, “delaying”the development of a disease (such as cancer) means to defer, hinder,slow, retard, stabilize, and/or postpone progression of the disease.This delay can be of varying lengths of time, depending on the historyof the disease and/or individuals being treated. A method that “delays”or alleviates the development of a disease, or delays the onset of thedisease, is a method that reduces probability of developing one or moresymptoms of the disease in a given time frame and/or reduces extent ofthe symptoms in a given time frame, when compared to not using themethod. Such comparisons are typically based on clinical studies, usinga number of subjects sufficient to give a statistically significantresult.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of cancer includes initial onset and/orrecurrence.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the agent to the subject, dependingupon the type of disease to be treated or the site of the disease. Thiscomposition can also be administered via other conventional routes,e.g., administered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesional,and intracranial injection or infusion techniques. In addition, it canbe administered to the subject via injectable depot routes ofadministration such as using 1-, 3-, or 6-month depot injectable orbiodegradable materials and methods.

Combination Therapies

In some embodiments, the methods described herein comprise administeringan agent described herein in in conjunction with an additional therapy.In some embodiments, the additional therapy is an immunomodulatoryagent. In some embodiments, the additional therapy is an agent thatinhibits cancer cell growth.

Immunomodulators

In any of the foregoing aspects and following embodiments, the one ormore immune checkpoint inhibitors are, for example, each an antagonistof programmed death 1 (PD-1), programmed death ligand 1 (PD-L1),cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), 15 V-domain Igsuppressor of T cell activation (VISTA), programmed death ligand 2(PD-L2), indoleamine 2,3-dioxygenase (IDO), arginase, B7 familyinhibitory ligand B7-H3, B7 family inhibitory ligand B7-H4, lymphocyteactivation gene 3 (LAG3), 2B4, B and T lymphocyte attenuator (BTLA), Tcell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2areceptor (A2aR), a killer inhibitory receptor, and/or signal transducerand activator of 20 transcription (STAT)3. In particular embodiments,the one or more immune checkpoint inhibitors are each an antagonist ofprogrammed death 1 (PD-1), an antagonist of programmed death ligand 1(PD-L1), an antagonist of cytotoxic T-lymphocyte-associated antigen 4(CTLA-4), and/or an antagonist of V-domain Ig suppressor of T cellactivation (VISTA).

In any of the foregoing aspects and embodiments, the PD-1 antagonist is,for example, an 25 agent that binds to and antagonizes PD-1. Such agentscan be, for example, a peptide that binds PD-1. Such agents can be ahumanized antibody that selectively binds PD-1. In some embodiments, thehumanized antibody that selectively binds PD-1 is nivolumab,pembrolizumab, pidilizumab, MEDI-0680, REGN2810, or AMP-224. In someembodiments, the humanized antibody that selectively binds PD-1 isnivolumab, pembrolizumab, or 30 pidilizumab. In some embodiments, theantagonist is (i) an antisense molecule directed against PD-1 nucleicacid, (ii) an adnectin directed against PD-1 nucleic acid, (iii) asingle stranded or double stranded RNAi inhibitor of PD-1, and/or (iv) asmall molecule inhibitor of PD-1.

In any of the foregoing aspects and embodiments, the PD-L1 antagonistis, for example, an agent that binds to and antagonizes PD-L1. Suchagents can be, for example, a peptide that binds PD-L1. Such agents canbe a humanized antibody that selectively binds PD-L1. In someembodiments, the humanized antibody that selectively binds PD-L1 isBMS-936559/MDX-1105, MPDL3280A/RG7446/atezolizumab,MSB0010718C/avelumab, or MEDI4736/durvalumab. In some embodiments, theantagonist is (i) an antisense molecule directed against PD-L1, (ii) anadnectin directed against PD-L1, (iii) a single stranded or doublestranded RNAi inhibitor of PD-L1, or (iv) a small molecule inhibitor ofPD-L. 10 In any of the foregoing aspects and embodiments, the CTLA-4antagonist is, for example, an agent that binds to and antagonizesCTLA-4. Such agents can be, for example, a peptide that binds CTLA-4.Such agents can be a humanized antibody that selectively binds CTLA-4.In some embodiments, the humanized antibody that selectively bindsCTLA-4 is ipilimumab or tremelimumab. In some embodiments, the CTLA-4antagonist is (i) an antisense molecule directed against CD80, CD86,and/or CTLA-4 nucleic acid, (ii) an adnectin directed against CD80,CD86, and/or CTLA-4 nucleic acid, (iii) a single stranded or doublestranded RNAi inhibitor of CD80, CD86, and/or CTLA-4, or (iv) a smallmolecule inhibitor of CD80, CD86, or CTLA-4.

In any of the foregoing aspects and embodiments, the VISTA antagonistis, for example, an agent that binds to and antagonizes VISTA. Suchagents can be, for example, a peptide. Such agents can be an inhibitoryantibody directed to VISTA. In some embodiments, the agent that binds toand antagonizes VISTA is a humanized antibody. In some embodiments, theagent that binds to and antagonizes VISTA is (i) an antisense moleculedirected against VISTA nucleic acid, (ii) an adnectin directed againstVISTA nucleic acid, (iii) a single stranded or double stranded RNAiinhibitor of VISTA, or (iv) a small molecule inhibitor of VISTA.

Chemotherapeutic Agents

In some embodiments, the methods described herein comprise administeringan agent described herein in in conjunction with an agent that inhibitscancer cell growth.

In some embodiments, an agent that inhibits cancer cell growth comprisesa chemotherapeutic agent. Chemotherapeutic agents include, for example,including alkylating agents, anthracyclines, cytoskeletal disruptors(Taxanes), epothilones, histone deacetylase inhibitors, inhibitors oftopoisomerase I, inhibitors of topoisomerase II, kinase inhibitors,nucleotide analogs and precursor analogs, peptide antibiotics,platinum-based agents, retinoids, vinca alkaloids and derivativesthereof. Non-limiting examples include: (i) anti-angiogenic agents(e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitorsof metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment),angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF solublereceptor, transforming growth factor beta, interferon alpha, soluble KDRand FLT-1 receptors, placental proliferin-related protein, as well asthose listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or aVEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants,soluble VEGF receptor fragments, aptamers capable of blocking VEGF orVEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosinekinases and any combinations thereof; and (iii) chemotherapeuticcompounds such as, e.g., pyrimidine analogs (5-fluorouracil,floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs,folate antagonists and related inhibitors (mercaptopurine, thioguanine,pentostatin and 2-chlorodeoxyadenosine (cladribine));antiproliferative/antimitotic agents including natural products such asvinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubuledisruptors such as taxane (paclitaxel, docetaxel), vincristine,vinblastine, nocodazole, epothilones, and navelbine,epidipodophyllotoxins (etoposide and teniposide), DNA damaging agents(actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethyhnelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) andgrowth factor inhibitors (e.g., fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin, mitoxantrone, topotecan, and irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisone, and prednisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

In some embodiments, an agent that inhibits cancer cell growth comprisesexternal radiation therapy and internal radiation therapy (also calledbrachytherapy). Energy sources for external radiation therapy includex-rays, gamma rays and particle beams, energy sources used in internalradiation include radioactive iodine (iodine125 or iodine131),strontium89, or radioisotopes of phosphorous, palladium, cesium, indium,phosphate, or cobalt. Methods of administering radiation therapy arewell known to those of skill in the art.

In some embodiments, an agent that inhibits cancer cell growth comprisesa CD-44 antagonist. In some embodiments, the CD-44 antagonist ishyaluronic acid. In some embodiments, the CD-44 antagonist is ananti-CD-44 antibody.

In some embodiments, an agent that inhibits cancer cell growth comprisesa CD133 antagonist.

In some embodiments, the methods described herein comprise administeringan agent described herein in in conjunction with an agent that is anantagonist of inhibitory macrophages and/or dendritic cells.

Screening methods

In some embodiments, the methods described herein provide foridentifying a test compound that will convert a Treg into a Teff. Themethod comprises:

-   -   contacting a regulatory T cell (Treg) with a test compound;    -   measuring IL-23R activity in the cell;    -   measuring IL-12Rβ activity in the cell;    -   wherein the test compound is a compound for converting a        regulatory T cell (Treg cell) into an effector cell if        (i) the IL-23R activity is decreased relative to a control cell        that has been treated with a compound known to not decrease        IL-23R activity and IL-12Rβ activity; and        (ii) the IL-12Rβ activity is not decreased relative to the        control cell.

In some embodiments, contacting a regulatory T cell (Treg) with a testcompound comprises contacting a regulatory T cell (Treg) with a testcompound in vitro using the methods for in vitro differentiationdescribed herein.

In some embodiments, IL-23R activity in the cell is measured. In someembodiments, measuring IL-23R activity comprises measuring expression ofFoxP3, Helios, IL-17A, IL-17F, ROR-gamma, and/or IL-23. In someembodiments, measuring IL-23R activity comprises measuring expression ofinterferon-gamma. In some embodiments, measuring IL-23R activitycomprises measuring STAT3 phosphorylation.

In some embodiments, a decrease in IL-23R activity comprises a decreasedin IL-23R activity relative to a control as described supra. In someembodiments, a decrease in IL-23R activity comprises a decrease inexpression of FoxP3, Helios, IL-17A, IL-17F, ROR-gamma, and/or IL-23, asis described supra.

In some embodiments, IL-12Rβ activity in the cell is measured. In someembodiments, measuring IL-12Rβ activity comprises measuring expressionof interferon-gamma.

In some embodiments, IL-12Rβ activity does not decrease if IL-12Rβactivity does not decrease relative to a control as described supra. Insome embodiments, IL-12Rβ activity does not decrease if IL-12Rβ2,IL18-Rβ1, IRF1, IL-2R, and/or G6NT expression does not decrease asdescribed supra.

In some embodiments, the test compound is a polypeptide, a nucleic acid,or a small molecule, as is described herein.

In some embodiments, effector cytokines produced by the cell aremeasured. Effector cytokines include, but are not limited to TNF-α,IFN-γ, IL-17, IL-10, or IL-2.

EXAMPLES Example 1: IL-23R as a Target in T Regulatory (Treg) Conversionand Anti-Tumor Therapy

Immunological approaches to the treatment of cancer have shown greatpromise for increased long-term survival for a number of cancers andcomplete remission in others. The number of clinical trials usingimmunotherapy alone or in combination with conventional cancertreatments has grown remarkably, based on increased understanding of thecritical contribution of T cell subsets to the control of cancer growth.

Regulatory T cells are critically important for maintenance of abalanced immune system, in part because they help to dampen excessiveimmune responses and prevent autoimmunity. However, immune suppressionexerted by Treg can impede anti-tumor immune responses and promotecancer growth. The efficacy of several successful immunotherapies hasbeen associated with depletion or blockade of CD4 regulatory T-cells(Treg) (Mahoney et al., Nat Rev Drug Discov 14:561-584). Described inthis example is a more advanced strategy that depends on selectiveinduction of Treg phenotypic instability and effector cell conversion ofintratumoral but not systemic Treg.

Impact of Helios Regulated STAT5 Activation on nTreg Stability

It was previously determined that the Helios transcription factor (TF)maintains stable expression of FoxP3 under inflammatory conditions viaregulation of STAT5b (Mahoney et al., Nat Rev Drug Discov 14:561-584).However the precise mechanism of Helios-dependent Treg stability andfunction has not yet been fully clarified. The data provided belowdefines the molecular basis of Helios-mediated stability and thebiological activity of conventional Treg in the tumor microenvironment(TME).

Helios was recently defined as a critical transcriptional factor thatensures stable expression of the Treg phenotype, which shows thatinhibition of Helios expression in intratumoral CD4+ Treg inducesreprogramming of Treg into T-effector cells that can destroy tumor cells(Nakagawa et al., Proc Natl Acad Sci U S A 113:6248-6253, Kim et al.,Science 350:334-339). The response is marked by decreased numbers ofintratumoral CD4 Treg, increased conversion of intratumoral Treg to Teffector cells and increased production of effector cytokines by CD8 Tcells. Since Treg conversion is restricted to intratumoral CD4 Treg,potential systemic toxicity and IRAEs may be avoided.

It has recently been shown that Helios^(−/−) nTreg exhibit reducedexpression of T cell anergy markers CD73 and FR4 which correlated withincreased proinflammatory cytokine expression under inflammatoryconditions (Kim et al., Science 350:334-339). Using Helios^(−/−) micewith solid tumors (B16/F10 melanoma and MC38 colorectal cancer) as asource of inflammation, nTreg anergic phenotype was characterized (FIGS.1A and 1B). nTreg loss of function was highlighted by reduced tumor size(FIG. 1A) and increased cytotoxic T cell activity (FIG. 1B).

Chromatin immunoprecipitation DNA sequencing (ChIP-seq) analysis of Tregcells indicated that the STAT5b locus contains multiple Helios bindingregions including a prominent Helios binding region located within the5′ enhancer intron of STAT5b (illustrated in FIGS. 2A-B). It is possiblethat Helios-dependent STAT5b interaction enhances Stat5b expression andstabilizes the nTreg phenotype via binding to the FoxP3 conservednoncoding sequence 2 (CNS2) region. CRISPR/Cas9 genome editing can beused to delete the putative Helios binding sites on STAT5b and generategenetically modified mice in which the interaction between Helios andStat5b is interrupted.

Immunological Activity of Converted Th1-Like Cells

Selective deletion of Helios in FoxP3⁺ Treg was found to result indecreased Treg-dependent inhibition of an anti-tumor response in B16/F10inoculated Rag2^(−/−) reconstituted mice (2×10⁶ CD4: 1×10⁶ CD8: 0.5×10⁶Helios^(WT) or Helios^(KO) nTreg) (FIG. 3A) which correlated withreduced FoxP3 expression (FIG. 3B) and increased production of thepro-inflammatory cytokine IFN-γ (FIG. 3C). Interestingly, the reducedFoxP3 expression and increased IFN-γ production by Rag2^(−/−)Helios^(f1/f1) FoxP3^(Cre) mice was only noted in the tumor infiltrate(FIGS. 3B-3C). No significant changes were observed in spleen.

Helios deletion was also found to specifically destabilize Tregs in asecond tumor model (MC38 colorectal cancer cell line) within the tumormicroenvironment (FIGS. 4A and 4B). Loss of the Treg-mediatedsuppressive function (FIG. 4 ) resulted in decreased FoxP3 expression(FIG. 4A) as well as anergy markers CD73 and FR4 (FIG. 4B) suggestingthat nTreg stability in solid tumors is Helios dependent. Furthermore,TCR activation of nTreg under inflammatory conditions (anti-CD3,anti-CD28, IL-2, IL-4) was found to reduce the proportion of nTreg cellspositive for Helios and the surface receptor TIGIT (FIGS. 5A and 5B).Helios nTreg cells were decreased by nearly 50% (FIG. 5A) whenco-cultured with IL-4 and Helios⁺ TIGIT⁺ cells by ˜50% (FIG. 5B) withincreased IL-4 levels.

Antibodies that Induce Treg to Teff Conversion

Two major signaling pathways maintain Treg stability within the tumormicroenvironment (TME): 1) a Helios-dependent pathway that enhancesIL-2R responsiveness and STAT5 activation and/or 2) an IL-23R signalingpathway that activates STAT5 within intratumoral Treg (FIG. 6 ).Blockade of either one of these signaling pathways induces Tregconversion as described below.

FIGS. 7A-7D shows antibodies found to induce conversion of Treg→Teff.Anti-IL-23R antibody treated Tregs showed decreased FoxP3 and increasedIFN-γ expression compared to controls (FIGS. 7A-7D).

Converted Th1-Like nTregs Mediate Stronger Antitumor Activity

Isolated pure populations of homogeneous CD4 Treg cells were tested fordifferentiation into CD4 Teff cells after antibody engagement of Tregtarget receptors that included TNFRSF members (anti-GITR, anti-OX-40,anti-4-1BB) and others (e.g., anti-CD47 and anti-Nrp-1). This initialanalysis of isolated CD4 Treg indicated that engagement of GITR byantibodies in the presence of IL-4 leads to a Treg→Teff conversionassociated with downregulation of FoxP3 and Helios and upregulation ofIFNγ production (FIG. 8 ).

The GITR agonist, DTA-1, has been suggested to alter the stability ofTreg cells. An in vitro assay was used to test if loss of stabilityresulted in Treg conversion to Th1-like effector cells. FIGS. 9A and 9Bshows data from an experiment in which sorted C57BL/6 (WT) Treg (>90%pure CD4+CD25+) were cultured with plate bound anti-CD3/anti-CD28 in thepresence of IL-2 and IL-4 for 24 hours or 5 days, and then assessed forconversion defined as loss of FoxP3 and gain of IFN-γ expression. Inthis system, GITR agonist DTA-1 showed loss of FoxP3 and increase inIFN-γ expression (FIGS. 9 and 9B). It can be seen that when the Ab wasused either prophylactically or therapeutically, engagement of GITR onTregs by anti-GITR Ab (DTA-1) led to reduction in tumor size compared tothe negative control (FIG. 9A). Anti-GITR also led to an effectorphenotype in tumors, but not in the spleen, as shown in FIGS. 10B and10C.

Treg Conversion by Anti-IL-23R Antibody

Blocking of IL-23R signaling in Treg under inflammatory conditions(IL-2⁺IL-4⁺) was found to induce Treg conversion as judged from reducedexpression of FoxP3 and production of IFN-γ(FIGS. 11A and 11B). As canbe seen in FIG. 12 , like DTA-1, anti-IL-23R antibody lowers theexpression of FoxP3 and increases the expression of IFN-γ in Tregsplated with plate bound anti-CD3/anti-CD28 and cultured in the presenceof IL2 and IL4. This effect was found to be dependent on the dose ofanti-IL-23R antibody (FIG. 13 ).

Moreover, unlike other anti-IL-23R antibodies which fail to induceconversion (not shown), the anti-IL-23R antibody with conversionactivity recognizes the extracellular domain of the IL-23R chain ratherthan the IL-12Rβ common chain that also binds to IL-12p40 (FIG. 14 ).

IL-23R Expression

In terms of being able to target tumors specifically, it is relevant andimportant to note that Treg from tumors express high levels of IL-23R,while splenic Treg were found not to do so (FIG. 15 ). IL-23R expressionis significantly increased in Treg found in the tumor and tumor draininglymph node (TdLN) compared to the spleen (FIG. 16 ). It was also foundthat effector Treg (eTr) express high levels of IL-23R (FIG. 17 ). Tumorinfiltrating CD69⁺Icos⁺ also express high IL-23R (FIG. 18 ). FIG. 20shows that Helios Treg express high levels of IL-23R. Indeed, it wasfound that IL-23R expression is reduced in cells in which Helios isknocked out (FIG. 20 ), demonstrating the dependence of IL-23Rexpression on Helios.

IL-23R Regulates FoxP3 and IFN-γ Expression

In an in vitro experiment in which sorted Treg were plated with platebound anti-CD3/anti-CD28 and cultured in IL-2 and IL-4 for 5 days, itwas found that FoxP3 expression is increased after treatment withrecombinant IL-23 protein and then somewhat decreased in cells treatedwith recombinant IL-23 protein and p(40)₂ (FIG. 21 ). Conversely, theIFN-γ levels increased after treatment with recombinant IL-23 proteinand p(40)₂ (FIG. 21 ).

Anti-IL-23R Antibody Effects on FoxP3 and IFN-γ Expression

Data in FIG. 22 shows that blocking IL-23R ligation by eitheranti-IL-23R antibody or P(40)₂ must occur after activation of thereceptor to reduce FoxP3 expression. FIG. 23 also shows that blockingIL-23R ligation likely occurs after activation to induce IFN-γexpression in FoxP3^(lo) cells.

Anti-Tumor Activity of Anti-IL-23R Antibody In Vivo

Tumor outgrowth was observed to be delayed in MC38 inoculated C57BL/6mice treated with (p40)₂ homodimer (FIG. 24 ).

FIGS. 25A-25E show results from an experiment in which C57BL/6 mice wereinoculated with MC38 cancer cells subcutaneously and thereafteradministered anti-IL-23R antibody (or isotype control) before assessmentof Treg subsets and IL-23R expression in various immune cell subsets(FIG. 25A). Growth curves for individual mice are shown in FIG. 25B. Thegraph in FIG. 25C shows that the third mouse that was administeredanti-IL-23R antibody remained tumor-free. The data plotted as theaverage per treatment condition can be seen in FIGS. 25D and 25E.

Treatment of mice inoculated with MC38 cancer cells with anti-IL-23Rresulted in increased CD8 activity as indicated by IFN-γ expression(FIG. 26A) and induced intratumoral Treg conversion as indicated byincreased IFN-γ expression (FIG. 26B).

IL-23R expression was found to be significantly decreased in anti-IL-23Rtreated tumor and tumor infiltrating activated Treg (FIG. 27 ). It wasalso decreased in eTr found in the tumor (FIG. 28 ). These anti-Il-23Rtumor infiltrating Treg have decreased Helios expression (FIG. 29 ), andreduced intratumoral suppressive Treg compared to isotype-controltreated mice (FIG. 30 ). Anti-Il-23R antibody treatment of cancer cellsinoculated mice also increases CD8/Treg ratio and CD8 IFN-γ expression(FIG. 31 ).

SUMMARY

The data discussed in this example show that anti-IL-23R antibody thatrecognizes the extracellular domain of the IL-23R chain rather than theIL-12Rβ common chain that also binds to IL-12p40, converts Tregs toeffector type cells in the tumor microenvironment specifically.

Example 2: Identifying and Validating Antibodies that Induce TRegulatory (Treg) Conversion and Anti-Tumor Activity An In Vitro Assayto Measure Treg Conversion

Two major signaling pathways maintain Treg stability within the tumormicroenvironment (TME): 1) a Helios-dependent pathway that enhancesIL-2R responsiveness and STAT5 activation and/or 2) an IL-23R signalingpathway that activates STAT5 within intratumoral Treg (FIG. 6 ).Blockade of either one of these signaling pathways induces Tregconversion as described below. Based on this knowledge, an in vitroscreen was developed to identify target surface molecules expressed byTreg that induce Treg→Teff conversion after engagement by an antibody.Verification of the efficacy of antibodies that induce Tregreprogramming as judged by conversion of intratumoral Treg and enhancedanti-tumor immunity is performed in vivo thereafter.

The in vitro screening system allows identification of Treg surfacemolecules that, after engagement by antibodies, induce conversion ofTreg to T effector cells (FIG. 32 ). A reporter system that allowsdetection of a converted Treg phenotype by levels of fluorescenceintensity is used: red fluorescence protein (RFP) for FoxP3 and yellowfluorescence protein (YFP) for IFNγ is utilized. Isolated CD4 Treg(RFP⁺YFP⁺) are stimulated with anti-CD3/CD28 antibody in the presence ofIL-2 and IL-4 to mimic an inflammatory environment. The effect ofsurface molecule engagement by antibodies is tested in 96 well platesthat contain triplicates of test- and control antibody treated wells.Candidate surface molecules and Treg converting antibodies areidentified based on reduced RFP and increased YFP signals, whichindicate conversion of Treg to T effector cells. Candidate antibodiesthat display Treg reprogramming activity are validated individually invitro before testing them in vivo.

Use of Epitope Mapping to Identify Anti-IL-25R Treg ConvertingAntibodies

The anti-IL-25R antibody that is discussed in Example 1 is a ratanti-mouse antibody. Epitope mapping can be used to develop moreantibodies, including anti-human antibodies, that block IL-25R.

Binding sites of the rat anti-mouse IL-25R antibody on IL-25R are mappedusing standard epitope mapping techniques. Identification andcharacterization of the binding sites of anti-IL-25R antibody, which hasthe ability to block IL-25R and induce Treg conversion (see Example 1)is then used to design and develop new antibodies against IL-25R. Thisdata is also used to develop anti-human IL-23R antibodies. The sequenceof human IL-25R (SEQ ID NO. 1) is 67% identical to the sequence of mouseIL-23R (SEQ ID NO. 2). Since antibody binding sites are expected to beon the extracellular domain of IL-23R, sequence identity in only thatregion between species is used for further fine-tuning. The sequence ofextracellular domain of human IL-25R (SEQ ID NO. 1, underlined) is 70.5%identical to the sequence of the extracellular domain of mouse IL-23R(SEQ ID NO. 2, underlined).

Amino acid sequence of human IL-23R (extracellular domain is underlined):(SEQ ID NO. 1)MNQVTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMICTWNAGKLTYIDTKYVVHVKSLETEEEQQYLISSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPETVPQVTSKAFQHDTWNSGLIVASISTGHLISDNRGDIGLLLGMIVFAVMLSILSLIGIFNRSFRIGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNIGYKPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHENRISLLEKAmino acid sequence of mouse IL-23R (extracellular domain is underlined):(SEQ ID NO. 2)MKREREMRGFYYIWDMSHLTLQLHVVIALYVLFRWCHGGITSINCSGDMWVEPGEIFQMGMNVSIYCQEALKHCRPRNLYFYKNGFKEEFDITRINRTTARIWYKGFSEPHAYMHCTAECPGHFQETLICGKDISSGHPPDAPSNLTCVIYEYSGNMTCTWNTGKPTYIDTKYIVHVKSLETEEEQQYLASSYVKISTDSLQGSRKYLVWVQAVNSLGMENSQQLHVHLDDIVIPSASIISRAETINDTVPKTIVYWKSKIMIEKVFCfhktpetvpqvVHQTSQETGKRNWQPWSSPFVHQTSQTVSQVTAKSSHEPQKMEMLSATIFRGHPASGNHQDIGLLSGMVFLAIMLPIFSLIGIFNRSLRIGIKRKVLLMIPKWLYEDIPNMENSNVAKLLQEKSVFENDNASEQALYVDPVLTEISEISPLEHKPTDYKEERLTGLLETRDCPLGMLSTSSSVVYIPDLNTGYKPQVSNVPPGGNLFINRDERDPTSLETTDDHFARLKTYPNFQFSASSMALLNKTLILDELCLVLNQGEFNSLDIKNSRQEETSIVLQSDSPSETIPAQTLLSDEFVSCLAIGNEDLPSINSYFPQNVLESHFSRISLFQK 

Example 3: IL-23R Antibodies in Tumor Immunity

Tumor volume was measured in mice treated with IL-23R antibodies. Allmice were inoculated with MC38 cells (0.2×10⁶) and treated with isotypeor IL-23R antibodies (100 ug/mouse) given 3 times (i.p 100 uL everythird day after tumor formation). Both 12B2B6 (Biolegend clone) and258018 (R&D clone) antibodies were used. As is shown in FIG. 33 ,significant delay in tumor growth can be seen in the IL-23R (12B2B64)antibody treated group tumor volume (mm³). Mean±SEM *p<0.05.

Treg conversion was measured in mice treated with IL-23R antibodies.Sorted Treg (WT) were plated for 5 days with plate boundanti-CD3/anti-CD28 and in the presence of IL-2 (20 ng/mL) and IL-4 (50ng/mL). 12B2B64 and 258018 antibodies were added at 10 ng/mL on Day 0 ofculture. All samples were compared to Negative Control (no antibody).DTA-1 was used as a positive control for Treg conversion. FoxP3expression is shown in FIG. 34A and IFN-gamma expression is shown inFIG. 34B. Graphs show combined data from three experiments (mean±SEMANOVA p<0.05*p<0.01**p<0.001***p<0.0001****). IL-23R antibody(12B2B64-Biolegend) induces Treg conversion in vitro under inflammatoryconditions. Additional IL-23 receptor antibody (258018-R&D Systems) didnot show evidence of Treg converting (↓FoxP3 and ↑IFN-g) ability.

The eTr phenotype was also measured in intratumoral Treg in mice treatedwith IL-23R antibodies. All mice were inoculated with MC38 (0.2×10⁶) andtreated with isotype or 12B2B64 or 258018 IL-23R antibodies (100ug/mouse) given 3 times (i.p 100 uL every third day after tumorformation). Tumors were collected and digested on day 21 to extractinfiltrating immune cells. Cells were then assessed for activationmarkers without stimulation. Significantly reduced expression of IL-23R⁺effector Treg (eTr) (TCR⁺CD4⁺CD25⁺FoxP3⁺CD44⁺CD62L⁻IL-23r⁺) was observedin the Biolegend (12B2B64) antibody treated group. Representative dotplots shown in FIG. 35 were gated on Treg (TCR⁺CD4⁺CD25⁺FoxP3⁺) and eTre(TCR⁺CD4⁺CD25⁺FoxP3⁺CD44⁺CD62L⁻). Graphic analysis is based on IL-23R⁺eTr. Mean±SEM **p<0.01.

The pSTAT5:pSTAT3 ratio was also assessed in mice treated with IL-23Rantibodies. All mice were inoculated with MC38 (0.2×10⁶) and treatedwith isotype or 12B2B64 or 258018 IL-23R antibodies (100 ug/mouse) given3 times (i.p 100 uL every third day after tumor formation). Tumors andspleen were immediately fixed and assessed for STAT activation (pSTAT5and pSTAT3) without stimulation. Reduced expression of pSTAT5 wasobserved in the IL-23R (12B2B64) antibody treated group resulting in areduced overall pSTAT5/pSTAT3 ratio. Representative histograms aredepicted in FIG. 36 and ratios are shown.

Epitope mapping was done for the 12B2B64 and 258018 IL-23R antibodies.FIG. 37 shows an alignment of murine and human IL-23R and a schematicillustration of the domains of IL-23R. Using the proposed cytokinebinding domain, a peptide library constructed of 26 peptides. Thepeptides were 15 amino acids long with an additional 5 amino acids ofoverhang. Binding of the antibodies to the peptides was assessed viaELISA. The peptides were bound to a plate, the antibodies were incubatedwith the individual peptides at various concentrations, and the analysiswas completed in a plate reader. Of the 26 peptides in the library 23were not bound by either antibody. Antibody 12B2B64 boundWNAGKLTYIDTKYVV (SEQ ID NO. 3) and LTYIDTKYVVHVKSL (SEQ ID NO. 4).Antibody 258018 bound WNAGKLTYIDTKYVV (SEQ ID NO. 3), LTYIDTKYVVHVKSL(SEQ ID NO. 4), and KYLVWVQAANALGME (SEQ ID NO. 5). FIG. 38 shows theregions in the cytokine binding domain where the peptides are located.

Example 4: IL-23R Modulates Treg Conversion Via STAT5 and STAT3

Loss of IL-23R enhances Treg conversion generally and in tumor cellsspecifically. FIGS. 39A-39B show that loss of IL-23R enhances conversionof Treg→Teff generally in a Treg conversion assay. IL-23R^(KO) Tregsshowed decreased FoxP3 and increased IFN-γ expression compared toC57BL/6 controls (FIGS. 39A-39B). FIGS. 40A-40C show that loss of IL-23Renhances conversion of Treg→Teff in tumor cells and decreases tumorvolume.

Treg isolated from IL-23R^(KO) or C57BL/6 mice were FACS sorted andtransferred into Rag2^(−/−) mice via injection into the tail vein alongwith CD8⁺ T cells. The mice were then inoculated with MC38 cancer cellline and tumor volume, and FoxP3 and IFN-γ expression in Treg from TILand the spleen were measured. The results presented herein show thatloss of IL-23R decreases tumor volume, decreased FoxP3 expression andincreased IFN-γ expression compared to C57BL/6 controls. FIGS. 40A-40Cshow knockout of IL-23R enhances conversion of Treg→Teff in tumor cells.

Treatment of mice having tumors from inoculation with the EL4 cancercell line with anti-IL-23R antibody shows that treatment withanti-IL-23R antibody also enhances conversion of Treg→Teff in lymphomatumor cells. Briefly, mice were inoculated with EL4 cancer cells(0.4×10⁶) for 21 days before being treated with isotype or anti-IL-23Rantibody (100 μg/mouse) three times (i.p 100 μL every third day aftertumor formation). As shown in FIGS. 41A-41B, treatment of mice with EL4tumors with anti-IL-23R antibody decreased FoxP3 expression andincreased IFN-γ expression compared to controls. These results furtherdemonstrate that treatment with anti-IL-23R antibody enhances conversionof Treg→Teff in tumor cells.

To demonstrate that knockdown of IL-23R, via neutralizing antibody,impacts STAT5 and STAT3 expression levels, CD25⁺FoxP3⁺Tregs were treatedwith either isotype or anti-IL-23R antibody in vitro. As shown in FIGS.42A-C, CD25⁺FoxP3⁺ Tregs treated with anti-IL-23R antibody hadsignificant reduction in the pSTAT5:pSTAT3 ratio compared toisotype-treated. This resulted from a decrease in pSTAT5 expression andan increase in pSTAT3 expression in anti-IL-23R antibody-treated cellscompared to isotype-treated cells as determined by flow cytometry (FIGS.42A-42B). Furthermore, as shown in FIGS. 43A-43B, this significantreduction in pSTAT5 expression occurs shortly after treatment (54%pSTAT5 expression in anti-IL-23R antibody-treated compared to 63.8%pSTAT5 expression in isotype-treated at one hour) and persists on longertimescales (5.97% pSTAT5 expression in anti-IL-23R antibody-treatedcompared to 22.8% pSTAT5 expression in isotype-treated at five days).

To corroborate the effectiveness of anti-IL-23R antibodies to enhanceconversion of Treg→Teff in tumor cells via STAT5 and STAT3 expression, ain vivo model was tested with anti-IL-23R antibody. Similar to previousexperiments, mice were inoculated with MC38 cancer cells (0.2×10⁶),treated with isotype or anti-IL-23R antibody (100 μg/mouse) three times(i.p 100 μL every third day after tumor formation), and FACS analysiswas performed on Day 25 after inoculation. FIGS. 44A-44C show thattreatment of mice with MC38 tumors with anti-IL-23R antibody provided asignificant reduction in the pSTAT5:pSTAT3 ratio compared to micetreated with isotype, as anti-IL-23R antibody-treated mice had areduction in pSTAT5 expression and an increase in pSTAT3 expressioncompared to isotype-treated mice. Mice treated with anti-IL-23R antibodyalso had increased IFN-γ expression and decreased IL-23R expressionlevels compared to isotype-treated mice, as shown in FIGS. 44E-44F. FIG.44D demonstrates that anti-IL-23R antibody treatment decreases thenumbers of CD25⁺FoxP3⁺Tregs, compared to isotype treatment. In a similarexperiment, mice were inoculated with EL4 cancer cells (0.4×10⁶),treated with isotype or anti-IL-23R antibody (100 μg/mouse) three times(i.p 100 μL every third day after tumor formation), and FACs analysiswas completed 21 days after inoculation. As shown in FIGS. 45A-45B,treatment of mice with anti-IL-23R antibody decreased pSTAT5 expressionand provided a reduction in the pSTAT5:pSTAT3 ratio, compared toisotype-treated mice.

The role of STAT3 in tumor growth was further investigated.STAT3^(WT)Treg mice and STAT3^(KO)Treg mice were inoculated with MC38cancer cells (0.2×10⁶). STAT3^(KO)Treg mice have been geneticallyengineered for knockout of STAT3 in Tregs (STAT3^(f1/f1)FoxP3^(cre)).FIG. 46A shows that tumor growth, as assessed by tumor volume,progresses more slowly in STAT3^(KO)Treg mice then it does inSTAT3^(WT)Treg mice. Additionally, STAT3^(KO)Treg mice have increasedexpression levels of CD69 and IFN-γ compared to STAT3^(WT)Treg mice inCD8 TIL (FIGS. 46B-46C).

It was next investigated whether the anti-IL23R antibody directlyinteracts with IL23-R Treg. This was first investigated by looking attumor volume in C57BL/6 and Rag2^(−/−) mice inoculated with MC38 cancercells (0.2×10⁶) for nine days before being treated with isotype oranti-IL-23R antibody (100 μg/mouse) three times (i.p 100 μL every thirdday after tumor formation). FIG. 47A shows that C57BL/6 mice treatedwith anti-IL-23R antibody had a reduction in tumor volume at the end ofthe treatment period compared to C57BL/6 mice treated with isotype.There was no difference observed for Rag2^(−/−) mice.

Whether the anti-IL23R antibody directly interacts with IL23-R Treg wasnext investigated by looking at tumor volume in mice in which isolatedTregs alone or in conjunction with CD8⁺ T cells were injected into thetail veins of Rag2^(−/−) mice. The mice were then inoculated with MC38cancer cells (0.2×10⁶) for nine days before being treated with isotypeor anti-IL-23R antibody (100 μg/mouse) three times (i.p 100 μL everythird day after tumor formation). FIG. 47B demonstrates that anti-IL-23Rantibody decreases tumor volume, compared to isotype, in Rag2^(−/−) micethat have been injected with Tregs alone or in conjunction with CD8⁺ Tcells.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1-31. (canceled)
 32. A method for treating cancer in a human subject,the method comprising: administering to the human subject an agent thatdecreases IL-23R and does not decrease IL-12Rβ activity, in an amounteffective to treat the cancer.
 33. The method of claim 32, wherein theagent comprises a nucleic acid or small molecule.
 34. The method ofclaim 33, wherein the nucleic acid comprises a DNA, RNA or nucleic acidanalogue.
 35. The method of claim 32, wherein the agent binds a nucleicacid expressing IL-23R.
 36. The method of claim 35, wherein the nucleicacid comprises an RNA interference (RNAi) inhibitor.
 37. The method ofclaim 36, wherein the RNAi inhibitor comprises a small interfering RNA(siRNA).
 38. The method of claim 37, wherein the siRNA comprises atleast 5 consecutive nucleic acids from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 19-31 or a sequence at least90% identical thereto.
 39. The method of claim 36, wherein the RNAiinhibitor comprises a short hairpin RNA (shRNA).
 40. The method of claim32, further comprising administering to the human subject animmunomodulatory agent.
 41. The method of claim 40, wherein theimmunomodulatory agent comprises an immune checkpoint inhibitor.
 42. Themethod of claim 41, wherein the immune checkpoint inhibitor is anantagonist of a molecule selected from the group consisting of PD-1,TIM-3, TIGIT, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR andLAG3.
 43. The method of claim 32, further comprising administering tothe human subject an agent that inhibits cancer cell growth.
 44. Amethod for inducing differentiation of a regulatory T (Treg) cell to aneffector T cell (Teff), comprising contacting the Treg cell with anagent that decreases IL-23R activity and does not decrease IL-12Rβactivity.
 45. The method of claim 44, wherein the agent comprises anucleic acid or small molecule.
 46. The method of claim 45, wherein thenucleic acid comprises a DNA, RNA or nucleic acid analogue.
 47. Themethod of claim 46, wherein the DNA, RNA or nucleic acid analogue bindsa nucleic acid expressing IL-23R.
 48. The method of claim 44, whereinthe contacting occurs in vitro.
 49. The method of claim 44, wherein thecontacting occurs in a human subject.